Sex-identification DNA markers are useful tools for sexing organisms that lack externally visible sexual dimorphism, and thus, they provide biological information for ecological and evolutionary studies. Tunas of the genus Thunnus (Scombridae), which comprises 8 species, lack sexual dimorphism of external morphology or coloration. In this study, we applied recently developed genotypic sex-identification markers for Pacific bluefin tuna to other tuna species to evaluate their effectiveness in sex identification. A sex-identification marker named 'primer pair II' demonstrated relatively high effectiveness in all tuna species, except southern bluefin tuna. Primer pair II was further tested in 209 albacore individuals collected during the scientific observer program onboard Japanese commercial long-line vessels, and it demonstrated robust performance for genotypic sex identification. The sex ratio of this albacore sample (1:1.4) significantly deviated from the expected 1:1 with the dominance of males, and the mean body size of males was higher than that of females. As all cross-species amplifications of the male-specific markers, except those for the southern bluefin tuna, were male-heterozygous polymorphisms, it is likely that a male-heterozygous sex-associated region exists in the Thunnus genome. The evolution of sex-determination systems in tunas was analyzed by ancestral state reconstruction, which showed that a common ancestor, before the evolution of the genus, possessed the male-heterozygous sex-associated genome region.