The coexistence of three sexual phenotypes (male, female and bisexual) in a single species, ‘trioecy’, is rarely found in diploid organisms such as flowering plants and invertebrates. However, trioecy in haploid organisms has only recently been reported in a green algal species,Pleodorina starrii. Here, we generated whole-genome data of the three sex phenotypes ofP. starriito reveal a reorganization of the ancestral sex-determining regions (SDRs) in the sex chromosomes: the male and bisexual phenotypes had the same “male SDR” with paralogous gene expansions of the male-determining geneMID, whereas the female phenotype had a “female SDR” with transposition of the female-specific geneFUS1to autosomal regions. Although the male and bisexual sex phenotypes had the identical male SDR and harbored autosomalFUS1,MIDandFUS1expression during sexual reproduction differed between them. Thus, the coexistence of three sex phenotypes inP. starriiis possible.