The diversity of sex determination systems in animals suggests that sex chromosomes evolve independently across different lineages. However, the present data on these systems is largely limited and represented mainly by bilaterian animals. Sex chromosomes and sex determination system based on cytogenetic evidence remain a mystery among non-bilaterians, the most basal animals. Here, we investigated the sex determination system of a non-bilaterian (Goniopora djiboutiensis) based on karyotypic analysis and identification of locus ofdmrt1, a known master sex-determining gene in many animals. Results showed that among the three isolateddmrtgenes,GddmrtCwas sperm-linked. Fluorescence in situ hybridization revealed that 47% of the observed metaphase cells contained theGddmrtClocus on the shorter chromosome of the heteromorphic pair, whereas the other 53% contained noGddmrtClocus and pairing of the longer chromosome of the heteromorphic pair was observed. These findings provided the cytogenetic evidence for the existence of the Y sex chromosome in a non-bilaterian animal and supports male heterogamety as previously reported in other non-bilaterian species using RAD sequencing. The Y chromosome-specificGddmrtCsequence was most homologous to the vertebratedmrt1, which is known for its role in male sex determination and differentiation. Our result on identification of putative sex chromosomes forG.djiboutiensismay contribute into understanding of the possible genetic sex determination systems in non-bilaterian animals.