Ribosomal coding DNA was sequenced and compared in 95 isolates of Mycosphaerella spp. collected in Nigeria and single nucleotide polymorphism (SNP) was used to identify the species and to determine the genetic structure of the sampled geographical populations. Using reference GenBank accessions with intercontinental distributions as controls, and shared species-specific SNPs in these control accessions, 84 (88·4%) isolates that grouped into 14 SNP haplotypes were identified as M. fijiensis, while 11 (11·6%) isolates represented by seven SNP haplotypes were characterized as M. eumusae. None of the isolates were either M. musicola or M. musae. The presence of M. fijiensis and M. eumusae in the collection was further confirmed using previously published species-specific probes designed on actin and β-tubulin gene sequences. A pairwise comparison of the population genetic distances revealed significant genetic differentiation between most populations (P < 0·001), with an average FST of 0·126, and a population structure corresponding to the four sampled geographical zones. The intraspecific dissimilarity of M. eumusae was 4·6%, compared with 2% for M. fijiensis. Compared to all the GenBank reference accessions, three sequence variations were unique to some Nigerian M. fijiensis haplotypes. Twenty-one sequence haplotypes were identified, geographically mapped and registered in GenBank. The results indicate that M. musicola has been replaced by more frequently occurring M. fijiensis and M. eumusae, against which disease management and resistance breeding efforts should be directed in Nigeria.