Operated by Yarra Trams, the route is coloured orange and extends from Box Hill to Port Melbourne over 19.3 kilometre of double track via Whitehorse Road, Victoria Street and Collins Street.

The origins of route 109 lie in separate tram lines, a cable tram from Spencer Street to the Yarra River, a horse tram from the Yarra River to Kew Cemetery, an electric line from Kew Junction to Box Hill (extended over the years), as well as the Port Melbourne railway line.

[1] A connecting horse tram was built from the east side of the Yarra River to Kew Cemetery, along Barkers Road, High Street South and High Street, opening on 28 December 1887.

[2][3] In 1929 the Melbourne & Metropolitan Tramways Board (MMTB) started converting the Collins Street cable lines to electric traction, with the last cable tram running down Collins Street on 14 September 1929, and the first electric tram on 8 December 1929.

[6][7][8] The broad gauge 1,600 mm (5 ft 3 in) track was re-gauged to standard gauge 1,435 mm (4 ft 8+1⁄2 in) and the overhead voltage was reduced from 1500 V DC to 600 V DC with light rail platforms built adjacent to or near the former stations platforms.

[17] The line was extended from its original terminus at Mont Albert to Box Hill along Whitehorse Road for 2.2 kilometres on 2 May 2003.