The mechanism of temperature adaptation in plants, including the formation of polyunsaturates in seed storage lipids, most likely involves transcriptional as well as post‐translational regulation of fatty acid desaturase activity. The present investigation was conducted to measure changes in the transcript accumulation among the three members of the soybean [Glycine max (L.) Merr.] microsomal omega‐3 fatty acid desaturase gene family in response to altered growth temperature during seed development. Microsomal omega‐3 fatty acid desaturases catalyze the insertion of a third double bond into linoleic (18:2Δ9, 12) acid to produce linolenic (18:3Δ9, 12, 15) acid. At 35 d after flowering, transcript accumulation (normalized for soybean actin) of GmFAD3A decreased by 5‐ to 15‐fold, GmFAD3B by 2‐ to 9‐fold, and GmFAD3C by 2‐ to 3‐fold in seeds that developed in a warm (day/night [D/N] = 30/26°C) versus a normal (D/N = 26/22°C) or a cool (D/N = 22/18°C) environment. At this stage of seed development, decreased omega‐3 desaturase gene expression levels were positively associated with reductions of 39 to 50% in the linolenic acid content of seeds of three soybean varieties examined. Thus, transcriptional regulation of the microsomal omega‐3 fatty acid desaturase gene family likely accounts, at least in part, for the reduced linolenic acid levels in soybean seeds grown at elevated temperature.