The fatty acid profile of the galaxiid fish Galaxias maculatus (Salmoniformes) collected from an estuarine habitat (a river mouth), a freshwater creek and two land-locked freshwater lakes in Victoria, Australia, were investigated with a view to evaluating habitat influences on the fatty acid profile. Fish from the freshwater creek were unpigmented, fresh-run juveniles (referred to as whitebait), the others adult fish. The fatty acids that predominated in adult G. maculatus muscle were 16:0 and 22:6(n-3), and 16:0 and 18:1(n-9), respectively. In whitebait 22:6(n-3), 20:5(n-3) and 16:0 were predominant. Hopkins estuary fish had the highest amount of 18:2(n-6) but the lowest 18:3(n-3). In all galaxiid populations, polyunsaturated fatty acids (PUFA) were the main group of fatty acids in total lipid in muscle, and accounted for more than 40 %, followed by saturates and monoenes. In landlocked populations, the amount of monoenes was lower than in other galaxiid populations. G. maculatus whitebait had significantly lower amount of saturates and the highest amount of n-3 fatty acids. The ratio of n-3 to n-6 PUFA ranged from 4.6 (Hopkins estuary) to 7.6 (whitebait), and the former differed significantly from all the other populations. The main fatty acids predominating in the liver of galaxiids from landlocked (Lake Bullen Merri) and estuarine populations were 16:0, 22:6(n-3) and 18:1(n-9), and generally reflected the corresponding pattern in muscle. A principal component analysis of the fatty acid composition of muscle samples confirmed and highlighted the distinct fatty acid profile of the populations investigated, when three groupings could be discerned. The study suggests that in addition to the diet, other habitat related factors may influence the fatty acid profile in catadromous G. maculatus.