Transgene stability in doubled haploid (DH) lines is an important consideration for transgenic cultivar development programs. The objective of this study was to assess the expression stability of acyl-acyl carrier protein (ACP) thioesterase (TE) transgenes in oilseed rape (Brassica napus L.) DH lines. The DH lines were developed from microspores of F1 plants from the crosses between transgenic parents carrying the bay-TE (Uc FatB1), elm-TE (Ua FatB1), nutmeg-TE (Mf FatB1), or cuphea-TE (Ch FatB1) transgenes and three nontransgenic cultivars having distinct seed oil fatty acid compositions. Of 333 DH1 plants developed from microspore-derived embryos that had undergone selection for kanamycin resistance (the selectable marker) with bay-TE F1 and cuphea-TE F1 plants as the microspore donors, 20 plants did not show TE transgene expression. Polymerase chain reaction (PCR) and Southern blotting analyses confirmed that the absence of TE transgene expression in these 20 DH1 plants was due to escape of embryos from kanamycin selection or existence of an incomplete T-DNA copy without the TE transgene. No DH1 plant with completely silenced TE transgenes was detected. Thirty of 34 transgenic DH lines showed a stable level of the target fatty acid, lauric acid (C12:0) for the bay-TE and palmitic acid (C16:0) for the other three TE transgenes, over the two or three consecutive generations tested (DH2, DH3, and/or DH4 plants). Target fatty acids were significantly affected by temperature during seed development. DH lines carrying the elm-TE or the cuphea-TE transgene grown under high temperature conditions (25/20°C, day/night) during seed development showed higher levels of palmitic acid than under lower temperatures (20/15°C). These results support the application of the DH procedure in breeding programs for transgenic cultivars and indicate the important influence of environment.