Flathead mullet (Mugil cephalus) is a cosmopolitan mugilid species popular in fishery and aquaculture with an economic preference for all-female population. However, it displays neither sexual dimorphisms nor heteromorphic sex chromosomes. In our previous study, we have generated the first draft of linkage map of the flathead mullet locating a single sex determination region (SDR) on its linkage group 9 (LG9) with evidence for XX/XY sex determination (SD) mechanism. In this work, the main objective was to refine the SDR on LG9 and propose positional candidate genes for SD based on expression and function. To elucidate the genetic mechanism of SD, we assembled and compared male and female genomic sequences of 19 syntenic genes within the putative SDR on mullet’s LG9, based on orthology to tilapia’s LG8 (tLG8) physical map. A total of 25 sequence-based markers in 12 genes were developed. For all markers, we observed association with sex in at least one of the two analyzed M. cephalus full-sib families, but not in the wild-type population. Recombination events were inferred within nuclear families setting the boundaries to an SDR orthologous to ~0.9 Mbp with 27 genes on tLG8. As the realized sex of each larva will not be known until it reaches sexual maturity, larvae were assigned into two sex-groups following segregation of the paternal haplotypes, following a model of XY/XX SD-system. A total of 107 sex-biased differentially expressed genes in larvae were determined, of which 51 had ortholog genes mapped to tLG8 (48% enrichment), as compared to 5% in random control. Of the 27 genes in the SDR, DOCK1, DHX32A and BCCIP were attractive candidates considering theirs potential function previously associated with SD, and were sex-biased differentially expressed in larvae (11% enrichment). Functional and positional enrichment of genes on mullet’s LG9 critical region that determines sex suggests the possible role of these three genes in SD, and lends further support for males being heterogametic for this SDR.