We quantified the effects of temperature, light and nitrogen availability on the growth and fatty acids (FAs) in three isolates of the green seaweed Derbesia tenuissima to portion the environmental and the genotypic (between isolates) components of variation. Growth ranged from 13 to 33% day− 1 and 27% of the variation was between isolates and 48% of variation was explained by light intensity. The content of total FA (TFA) ranged from 34 to 55 mg g− 1 dw and 49% of the variation was between isolates, while the TFA was 20% lower in the high light and low nitrogen treatment combination. The proportion of omega-3 polyunsaturated FA (PUFA(n-3)) ranged from 31 to 46% of TFA with a strong interactive effect of isolate and temperature. In two isolates, the proportion of PUFA(n-3) increased by 20% under cultivation at low temperature while in a third isolate temperature had no effect. Increases in PUFA(n-3) occurred with a stable content of TFA and high growth rates, leading to net increases in PUFA(n-3) productivity in two isolates. This research highlights the potential for environmental manipulation and strain selection to further improve the productivity and quality of fatty acids in seaweed.