Dioecy has evolved recently and independently from cosexual populations in many angiosperm lineages, providing opportunities to understand the evolutionary process underlying this transition. Spinach ( Spinacia oleracea ) is a dioecious plant with homomorphic sex chromosomes (XY). Although most of the spinach Y chromosome recombines with the X chromosome, a region around the male-determining locus on Y does not recombine with its X counterpart, suggesting that this region might be related to the evolution of dioecy in the species. To identify genes located in the non-recombining region (MSY, male-specific region of Y), RNA-seq analysis of male and female progeny plants (eight each) from a sib-cross of a dioecious line was performed. We discovered only 354 sex-chromosomal SNPs in 219 transcript sequences (genes). We randomly selected 39 sex-chromosomal genes to examine the reproducibility of the RNA-seq results and observed tight linkage to the male-determining locus in a spinach segregating population (140 individuals). Further analysis using a large-scale population (>1400) and over 100 spinach germplasm accessions and cultivars showed that SNPs in at least 12 genes are fully linked to the male-determining locus, suggesting that the genes reside in the spinach MSY. Synonymous substitution rates of the MSY genes and X homologues predict a recent divergence (0.40 ± 0.08 Mya). Furthermore, synonymous divergence between spinach and its wild relative ( S . tetrandra ), whose sex chromosomes (XY) originated from a common ancestral chromosome, predicted that the species diverged around 5.7 Mya. Assuming that dioecy in Spinacia evolved before speciation within the genus and has a monophyletic origin, our data suggest that recombination around the spinach sex-determining locus might have stopped significantly later than the evolution of dioecy in Spinacia .