The Green Bay Route's first branch from the main line, and the first to be abandoned.

Construction of the La Crosse branch began in March 1876 and the first regular train operated on September 25, 1876. The branch was abandoned in 1922.

The Green Bay & Western operated 21.59 miles over the Chicago & North Western (C&NW), via a trackage rights agreement from Marshland to Onalaska using the ex- La Crosse, Trempealeau and Prescott RR's "Winona Cutoff." From where it left the C&NW, the final 6.38 miles from Onalaska to La Crosse was owned by the GB&W and the C&NW operated on the GB&W tracks. It was apparently a convenient way for the C&NW to enter La Crosse, which was a Milwaukee Road stronghold.

The original plan was to complete a line from Blair or Merrillan on the GB&W main line to La Crosse and then over the Mississippi River to an interchange with the Southern Minnesota Railway (SM). Only the La Crosse portion was ever built. For a brief time the La Crosse trackage served as the western terminus of the GB&W, with through-train service from Green Bay. This proved unprofitable and an improved connection between East Winona and Winona over the Mississippi River soon delegated the La Crosse tracks to branch line status.

The stations and mileposts on the La Crosse branch were:

From Onalaska, the GB&W tracks passed downgrade through several shallow cuts and crossed the Milwaukee Road and Chicago Burlington & Quincy at Grand Crossing. As a matter of fact, the Green Bay & Minnesota / Chicago, Milwaukee & St. Paul crossing was the first to be built at that location.  The line then went south over swampland on trestles that were filled in later years and entered La Crosse at the State Teachers College (later UW-La Crosse) campus. It skirted the city on the east and then curved sharply west, terminating near the tracks of the Chicago, Burlington & Quincy and La Crosse & Southeastern and opposite the SM's terminal in LaCresent, Minnesota.

The first GB&W La Crosse depot was shared with the C&NW at 9th Street and Green Bay Street. It consisted of a 80'x24' freight house, 26'x56' foot passenger building, and a 46'x214' foot train shed. The C&NW built its own line into the city and a depot in downtown La Crosse in 1886. In 1903 the GB&W built a 25 x 55 feet frame depot at 904 Green Bay Street. The GB&W ceased its La Crosse operations in 1922 and the depot was razed in 1973. The GB&W also owned property west of the terminal on which a Mississippi River bridge could have been built.

Much of the traffic on the branch consisted of coal loads for the State Teachers College and the local water works, gas, and electric companies. For the period from 1914-1920, the most important commodities hauled on the branch in terms of tonnage were 1) coal, 2) brick and stone, 3) merchandise, 4) beer, 5) lumber, 6) grain, 7) empty kegs, and 8) iron and steel.

The GB&W had an oppressive trackage rights agreement with the C&NW, which extracted high rent and limited them to only one mixed train per day. The branch lost money from 1885 onward; the GB&W filed for abandonment in 1921 but was opposed by the City of La Crosse, the Chamber of Commerce, and the State Railroad Commission. The GB&W then made a contract with the CB&Q giving access to the GB&W's customers and reapplied for abandonment in 1922, which was granted this time by the Interstate Commerce Commission. The last GB&W train operated on October 26, 1922.

The Green Bay Route retained actual ownership of the branch, with the CB&Q leasing 2.65 miles for $1.00 per year. Soon after becoming the president of the Green Bay Route, Homer McGee took action to sell the branch outright to the CB&Q. In July 1936 the sale was completed for $27,000.

Remnants of the line can be seen east of BNSF's North La Crosse yard and part of the grading of the line can be seen just north of UW-La Crosse.