Johnstons Creek, formerly Johnston's Creek, is an urban gully, located in Sydney, Australia and situated in the Inner West and Sydney local government areas. The creek flows from Petersham, past Annandale, Camperdown, Forest Lodge and Harold Park, before spilling into Rozelle Bay, within Sydney Harbour.

Originally a natural watercourse, Johnston's Creek was converted into a brick and concrete channel in the 1890s in order to improve sanitation in Sydney. The creek rises in Petersham and initially marked the eastern boundary of the land granted to Lieutenant-Colonel George Johnston in the 1790s, which he named Annandale. The Annandale Estate was subdivided in the latter part of the 19th Century into what is now the suburbs of Stanmore and Annandale. The channel now forms a boundary of Annandale, Forest Lodge, Camperdown and Stanmore.

Johnstons Creek has one minor tributary, Orphan School Creek, an urban canal that joins Johnstons Creek at Forest Lodge.

Johnstons Creek is crossed by the Annandale Bridge on Parramatta Road, and road bridges at Booth Street and The Crescent. The heritage-listed Allan Truss Bridge, formerly the Federal Road Bridge, crosses Johnstons Creek near its mouth, connecting Annandale and Glebe. The bridge was decommissioned for road usage and converted as a footbridge in 2000. A small pedestrian and road bridge crosses the canal at Harold Park, and a former tram bridge provides a direct route from Minogue Crescent to Hogan and Spindler Park. The Bowstring Bridge, a footbridge, is an early example of reinforced concrete bowstring arch bridges built in Australia, located on Minogue Crescent, Forest Lodge.

A heavy rail viaduct, now used for the Inner West Light Rail, crosses the creek between Jubilee Park and Federal Park, west of Glebe.

Completed in 1898, the Johnston's Creek Sewer Aqueduct connects Sydney's western suburbs to the Northern Main Sewer extension of the Bondi Ocean Outfall Sewer, managed by Sydney Water. The Johnston's and White's Creek Aqueducts, both listed on the Register of the National Estate, were the first reinforced concrete structures in Australia.

A salt marsh wetland was constructed in 2001 to filter storm water before it enters Sydney Harbour via Johnston's Creek.

In 2020 Sydney Water started a major project to naturalise the creek from The Crescent to the mouth at Rozelle Bay.

In September 2009 a bull shark believed to be 1.8 metres (5 ft 11 in) long was sighted in Johnstons Creek. The shark was last seen swimming along the Glebe foreshore in the direction of the Anzac Bridge.

• Whites Creek
• Glebe and Wentworth Park railway viaducts

• Johnstons Creek Stormwater Channel Exhibition)

34°36′S 150°35′E / 34.600°S 150.583°E / -34.600; 150.583