The purpose of this study was to identify maternal mRNA genes that are associated with reduced embryonic survival in rainbow trout (Oncorhynchus mykiss). Microarrays were used with mRNAs from unfertilized egg batches derived from fertile and sub-fertile females to determine differences in gene expression. Eggs from fertile females had 104 microarray targets that were significantly (p < 0.05) higher compared to those targets from sub-fertile females. Conversely, only six microarray targets were significantly (p < 0.05) higher in eggs from sub-fertile compared to fertile females. Five candidate genes were selected for real-time RT-PCR analysis to validate the microarray results. Only one gene, guanylate kinase (GK), corroborated the microarray analysis being consistently lower in eggs from sub-fertile females. This is the first maternal mRNA gene identified in rainbow trout that correlates with embryo survival. It is not known whether GK is directly or indirectly involved with egg viability, but it may be useful as a fertility biomarker in this fish.