The Panama route was divided into three different sections, namely, the Atlantic route from New York to Chagres, New Granada, the 60 mile route across the Isthmus of Panama from Chagres to Panama City and the Pacific route from Panama to San Francisco.

 
The United States Mail Steamship Company (USMSSCo.) obtained the contract for the New York to Chagres route. Calls were scheduled at various ports over the years, including Charleston, Savannah, Key West, Havana and New Orleans. Kingston, Jamaica was used as a coaling stop and mail was also picked up there. The service from New York was to begin October 1, 1848, but due to delays in the construction of steamers for the USMSSCo. a smaller steamer of 890 tons, the Falcon, was purchased and sailed from New York for Chagres on December 1, 1848. The two much larger steamships laid down for this service, the Ohio of 3,000 tons and the Georgia of 3,300 tons, sailed for Chagres on September 20, 1849, and January 28, 1850, respectively.

 
The overland route across the isthmus was at first carried out by the Pacific Mail Steamship Company (PMSSCo.) using mules and canoes to cross the isthmus. This service was quite unsatisfactory, often having delays. Late in 1849 the government of New Granada assumed responsibility for the carriage of mail across the isthmus, and they subcontracted the service to Panamanian firms. This did not turn out to be any improvement in the service.

Beginning January 1, 1852, the Panama Railroad (PRR) entered into a contract with the Post Office Department to carry the mails on a per pound basis. As the railroad neared completion this arrangement was changed to a flat rate of $100,000 per annum. The railroad was completed in late January of 1855, and the mail crossing of the isthmus became a short train ride of a few hours. After the ocean mail contracts expired June 30, 1860, the mail contract with the PRR was reduced to $25,000 for the following year, rising somewhat from this figure in later years.

The PMSSCo. ran their steamships between Panama City and San Francisco, with various calls at Acapulco, Manzanillo, San Blas, Mazatlan, San Diego and Monterey. After 1851, usually only one stop at either Acapulco or Manzanillo was made. The California was the first steamer built by the PMSSCo. and she was launched May 19, 1848. She sailed from New York for Panama, via Cape Horn, on October 6, 1848. The California arrived at Panama January 17, 1849 and steamed into San Francisco February 28, 1849. She was deluged with additional argonauts at Panama and upon arrival at San Francisco her entire crew save the Captain and one of the engine room boys deserted for the gold fields. The Panama was the second PMSSCo. steamer to be completed and she sailed from New York on December 1, 1848. Engine problems forced her back into port shortly thereafter and she did not leave again until February 15, 1849, arriving in San Francisco on June 4, 1849. The third PMSSCo. steamer, the Oregon, departed New York on December 9, 1848, arriving in San Francisco on April 1, 1849.

The mail contracts that began October 1, 1848, ran for ten years and were to expire on September 30, 1858. However, the Postmaster General and the Secretary of the Navy extended them for another year. At this time the USMSSCo. had withdrawn from the Panama route, and the PMSSCo., along with the PRR, formed the North Atlantic Steamship Company (NASS Co.), to operate a line between New York and Aspinwall. This line was to connect with the PMSSCo. ships in the Pacific.

In opposition to these companies Cornelius Vanderbilt had formed the Atlantic and Pacific Steamship Company (APSSCo.), with his ships in service on both sides of the continent. Both the PMSSCo. and Vanderbilt's APSSCo. bid on the mail contract, but it was awarded to a Daniel H. Johnson, a broker for other investors. When Johnson was unable to carry out the contract it was awarded to Vanderbilt. By February 1860, however, Vanderbilt and the PMSSCo. came to terms and in March the PMSSCo. resumed the carriage of mail on the Pacific side. This arrangement lasted until November of 1865 when the PMSSCo. took over the entire route from New York to San Francisco. After 1861 the compensation paid the steamship lines for the transportation of the mails was reduced considerably, it being subject to the amount of mails carried.

 
The first overland mail, the Butterfield route from St. Louis to San Francisco, left each city on September 15, 1858. This route signaled the decline of mail carriage on the Panama route. Large amounts of mail were still carried during the 1860's, and at times all of the mail was reverted to the Panama steamers because of Indian problems on the overland route. With the completion of the transcontinental railroad on May 10, 1869, the dominant position of the Panama route came to an end.