Species of the genus Diaporthe have a mating-type system with the two mating types MAT1-1 and MAT1-2, like other ascomycetes. They can either be heterothallic, which means that any isolate only possesses one of the two mating types and needs a mating partner for sexual reproduction, or homothallic, which means that they possess both mating types and are self-fertile. For several Diaporthe species, no sexual reproduction has been observed so far. Using PCR with primers specific to the defining genes MAT1-1-1 and MAT1-2-1, we determined the mating types of 33 isolates of Diaporthe caulivora, D. eres, D. longicolla, and D. novem from central Europe. In addition, we partially sequenced the mating-type genes of 25 isolates. We found that different D. longicolla isolates either possess MAT1-1-1 or MAT1-2-1, making the species heterothallic, which is in contrast to previous studies and the general assumption that D. longicolla only reproduces asexually. D. eres and D. novem were also found to be heterothallic. Using genomic sequence information and re-sequencing of DNA and RNA, we identified the MAT1-1-1 gene in D. caulivora and present here the full sequence of the mating-type locus of this homothallic species. Finally, we used sequence information from MAT1-1-1 and MAT1-2-1, respectively, for improved phylogenetic resolution of our isolates.