Dothistroma needle blight (DNB) is a serious disease of the Pinaceae, mainlyPinusspecies, caused by the fungiDothistroma septosporumandD. pini. Both species are regarded as invasive forest pathogens worldwide, with rising incidence in central and northern Europe over the last three decades. In this work, 29 sites were investigated between 2013 and 2015 in south-western Turkey. Morphological examination of needles confirmed DNB infection (i.e.,Dothistromaconidiospores observed) at 18 sites, and a total of 108Dothistromasp. isolates were obtained from 11 of the sites. Host age seemed to be an important factor in both occurrence and severity of DNB inPinus brutiaforests. Continuous rainy days, especially in December, may increase severity of disease; however, extreme rain events may reduce available conidiospores on plant tissues or in the air. Species-specific mating type primers showed that all isolates wereD. septosporum;D.piniwas not detected. The mating type ratio was close to 1:1, indicating sexual recombination was occurring. Eleven microsatellite markers revealed 59 unique multilocus haplotypes (MLHs) among the 73 isolates originating from different conidiomata. The majority of MLHs were represented by a single isolate (n = 52) and only one MLH was shared between two localities. Analyses showed high genetic diversity, isolation-by-distance, and clear population clusters. These findings suggest thatD.septosporumis well established in south-western Turkey and is probably not a recent introduction.