Allele frequencies were determined at 14 microsatellite loci in 284 female and 50 male rainbow trout that were sampled throughout the spawning season from a commercial trout farm. Phenotypic selection has expanded the spawning season of the broodstock from 2 weeks to 8 months. Females maturing in different seasons showed significantly different allelic distributions (P<0·001) at all loci. The spawning time for the majority of females sampled could be predicted based on their genotypic information [chromosome segment sharing coefficient (CSSC) values]. CSSC analyses assigned 100, 56, 76 and 68% of summer, fall, winter, and spring spawning females, respectively to the season from which their gametes were actually collected. Alternatively, only 38 and 14% of summer and spring spawning XY males respectively, were assigned to the correct season. Loci linked to thermal tolerance and spawning time quantitative trait loci (QTL) showed significantly greater heterogeneity (higher average Ds values) in allele frequencies than those not known to be linked to QTL based on previous work. Thus, phenotypic selection for spawning time has led to concomitant changes in allele frequencies at markers of QTL. This suggests that the QTL detected in our previous work have detectable effects in fish from other genetic backgrounds.