Brown rot disease caused by fungal species from the Monilinia genus has recently been observed as one of the most important limiting factors for yield and quality of peach fruits in Turkey. During June and July of 2018, field trips to different peach orchards were performed in six provinces located in four different geographical regions of Turkey. One hundred and twenty-nine isolates were obtained from the diseased fruits, and of those, 109 were identified as Monilinia fructicola, whereas 20 were M. laxa according to the species-specific molecular markers. Mating types of the isolates were detected by PCR assays using primers designed in this study. Each isolate represented only one of the mating type genes. Overall data sets presented 1:1 ratio of mating types for both species, indicating a possible sexual reproduction. No polymorphisms were detected in partial sequences of mating type genes. The isolates were in vitro tested for their pathogenicity using apple fruits as a host and aggressiveness were evaluated at different time points using lesion sizes. Monilinia laxa was found more aggressive than M. fructicola. Moreover, M. fructicola isolates displayed larger colony growth compared to M. laxa isolates. However, no significant correlation was detected between the colony growth rates and virulence. This study is the first to demonstrate brown rot pathogens of peach fruit in Turkey and identifies different aspects of the pathogens, which would be useful in containment of pathogen spread and comparison with other populations of these pathogens worldwide, and suggests a new set of mating type markers for these species.