Dothistroma needle blight (DNB) is a serious pine disease present worldwide caused by the ascomycetous fungi Dothistroma septosporum and Dothistroma pini. Based on multiplex PCR analysis of 11 microsatellite markers screened on 253 D. septosporum isolates obtained from 32 sites across Slovakia, a total of 137 unique multilocus haplotypes (MLHs) were detected. The majority of MLHs (n = 91) were represented by a single isolate, but 13 MLHs were found at two different sites, and in some cases, these were separated by large distances. Four population clusters were identified using a discriminant analysis of principal components (DAPC). The genetic clusters obtained from the DAPC were mixed throughout the country and were not restricted to particular host species. Although both mating types of D. septosporum were found to be in an almost perfect 1:1 ratio in both the non-clone-corrected and clone-corrected datasets, random mating was rejected in the entire dataset. Random mating was only supported in the Pinus mugo group planted in urban areas and on a smaller spatial scale in two sites (Kalnica and Litava) using the clone-corrected dataset. These findings suggest a mixed mode of reproduction with an important component of sexual reproduction, although the sexual stage of the fungus (teleomorph) has not been physically observed in Slovakia yet. The examination of spatial relationships using spatial principal component analyses and the presence of isolation-by-distance together with relatively high genetic diversity suggests the pathogen has been long established in Slovakia and spread naturally across the landscape. However, the weak population structure and findings of identical clones at widely separated sites strongly suggests some degree of human assisted dispersal.