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Pseudomonas syringae pv. syringae (Pss), which causes bacterial canker, is the most polyphagous bacterium in the P. syringae complex due to its broad host range. This pathogen is considered the major bacterial disease in cherry orchards. In this study, several samples were collected from infected sweet cherry trees in different locations of the Marmara region in Turkey between 2016-2018. Sixty-three isolates were identified as Pss by pathogenicity, LOPAT, GATTa, and MALDI-TOF MS tests. Total genomic DNA was extracted to confirm identity, followed by PCR amplification of syrB and cfl genes. Out of 63 isolates, 12 were randomly selected for Repetitive Element Sequence-based PCR (rep-PCR) and Multilocus Sequence Typing (MLST) analysis to gain insight into the relationships of those isolates. The cluster analysis of rep-PCR (ERIC-, REP- and BOX-PCR) could classify the isolates into two distinct clusters. Phylogenetic analysis was carried out to obtain the relation between isolates and the location.The MLST analysis of gyrB, rpoDp, rpoDs, and gltA genes allowed a clear allocation of the isolates into two separate main clusters. The relationship among the isolates were also evaluated by constructing a genealogical median-joining network (MJN). The isolates from six locations produced 11 haplotypes that were illustrated in the MJN. The results of this study proved that location could not be an indicator for showing the genetic diversity of Pss from cherry orchards. As the genetic variability of Pseudomonads has been demonstrated, the current study also showed high diversity among different isolates even within the populations. While more research is recommended, the results of this study contributed to a better understanding of the Pss evolutionary progress and genetic diversity of sweet cherry isolates.