Sexual reproduction of fungi is governed by genes located on the mating-type (MAT) locus. To analyze the MAT locus of the genus Diaporthe (anamorph: Phomopsis), a large genera within the ascomycetous class Sordariomycetes, we cloned and sequenced loci MAT1-1 and MAT1-2 from two heterothallic Diaporthe species, designated as Diaporthe W- and G-types (four isolates in total). The mating-type loci structures of Diaporthe W- and G-types were similar; MAT1-1 isolates had a MAT locus containing three genes, MAT1-1-1, MAT1-1-2 and MAT1-1-3, as was the case with other Sordariomycetes, and in contrast to other Sordariomycetes, MAT1-2 isolates had genes homologous to MAT1-1-2 and MAT1-1-3, in addition to MAT1-2-1. Expression analysis by RT-PCR revealed that all the mating-type genes of Diaporthe W-type were transcriptionally active during vegetative growth. The structure of MAT loci of Diaporthe W- and G-types is distinct from that in other heterothallic filamentous ascomycetes, which have dissimilar gene structure in opposite mating-type loci. This unique structure is informative to discussing the evolutionary history and function of mating-type genes of Sordariomycete fungi.