James Johnstone may refer to:
- James Johnstone, 1st Earl of Hartfell (1602–1653), Scottish peer and royalist
- James Johnstone, 2nd Marquess of Annandale (c. 1687–1730), Scottish peer
- Sir James Johnstone, 3rd Baronet (1697–1772), Scottish MP for Dumfries Burghs 1743–1754
- Sir James Johnstone, 4th Baronet (1726–1794), Scottish MP for Dumfries Burghs 1784–1790 and Weymouth & Melcombe Regis
- James Johnstone (explorer) (1759–1823), master, later lieutenant, aboard HMS Chatham during George Vancouver's 1791–95 expedition
- James Hope-Johnstone, 3rd Earl of Hopetoun (1741–1816), Scottish representative peer and Lord Lieutenant of Linlithgowshire
- Chevalier de Johnstone (1719–c. 1791), actually James Johnstone, Jacobite army officer
- James Johnstone (1801–1888), Scottish MP for Clackmannanshire & Kinross-shire
- James Johnstone (publisher) (1815–1878), British newspaper proprietor
- James Johnstone (biologist) (1870–1932), British biologist and oceanographer
- James Johnstone (stock breeder) (1859–1933), New Zealand businessman and stock breeder
- Jim Johnstone (poet) (born 1978), Canadian physiologist and poet
- Jim Johnstone (umpire) (1872–1927), American baseball umpire
- Jim Johnstone (basketball) (born 1960), American basketball player
- Jimmy Johnstone (1944–2006), Scottish footballer
- James Johnstone (rugby union) (born 1990), Scottish rugby union player