Dothistroma needle blight (DNB) is regarded as one of the most significant foliar diseases of pine and has caused significant damage to pine plantations in France since the late 1990s. The genetic diversity, reproductive strategy and population structure of D. septosporum, one of the causal agents of DNB, was investigated at six sites across Brittany, France, using species-specific mating type primers and microsatellite markers. Multiplex PCR assays were developed to effectively and efficiently screen 11 of the available microsatellite markers on the isolates obtained. From 282 isolates analysed, 81 unique multilocus haplotypes were identified, indicating a diverse (haplotypic diversity ranged from 0.403 to 0.962) yet highly clonal structure of the pathogen at these sites. Although all sites contained both mating types, random mating was rejected by the index of association and parsimony tree length permutation tests on both clone-corrected and non-clone-corrected datasets but supported by mating type ratios in the clone-corrected datasets. Four population clusters were identified using STRUCTURE. Neither locality nor host species played a significant role in the population structure in Brittany. These findings, along with the observation of identical haplotypes at different sites and on different hosts, suggest movement of the pathogen within the peninsula. Overall, the pathogen’s population structure was found to be influenced by sexual recombination yet within sites clonal reproduction predominated.