Many differential gene expression analyses are conducted with an inadequate number of biological replicates. We describe an easy and effective RNA-seq approach using molecular barcoding to enable profiling of a large number of replicates simultaneously. This approach significantly improves the performance of differential gene expression analysis. Using this approach in medaka (Oryzias latipes), we discover novel genes with sexually dimorphic expression and genes necessary for germ cell development. Our results also demonstrate why the common practice of using only three replicates in differential gene expression analysis should be abandoned.