We aimed to investigate proximate and fatty acid composition of important freshwater fish species in the Czech Republic. Sampled fish include seven species from intensive farming: African catfish, rainbow trout, Wels catfish, Nile tilapia, brook trout, northern whitefish, and pikeperch; eight species from semi-intensive culture systems: common carp, northern pike, pikeperch, grass carp, European perch, tench, silver carp, and catfish; and three species from extensive culture systems: rainbow trout, tench, and common carp. The fat content and fatty acid composition were highly influenced by the culture systems. Simultaneously, we observed a significant dependence of fatty acid composition on the fat content. The content of saturated fatty acids was below 34% in all analyzed fish. Northern pike, pikeperch, and European perch contained with over 50% the highest proportion of polyunsaturated fatty acids. Intensively cultured fish reached the highest content of eicosapentaenoic and docosahexaenoic fatty acid. Nutritional quality was determined by atherogenic and thrombogenic indexes which ranged from 0.27 to 0.63 and 0.20 to 0.61 and by ratios n-3/n-6 (0.54-3.45) and polyunsaturated/saturated fatty acids (0.67-2.01). Results demonstrated that the flesh of all studied species are of high nutritional quality.