Shot-hole disease is one of the most common and significant diseases affecting cherry trees in Gilgit-Baltistan, Pakistan, every year. It results in small reddish areas on leaves and fruits, forming characteristic holes. However, the pathogen associated with the disease, its pathogenicity, and its growth performance under different environmental conditions remains unknown. In this study, the pathogen associated with shot-hole disease was identified as W. carpophilus. Furthermore, variations in pathogenicity were observed among the five isolates of the pathogen (SRA-1, SRA-2, SRA-3, SRA-4, and SRA-5), with SRA-5 exhibiting smaller lesion sizes and SRA-1 to SRA-4 displaying larger lesion sizes. Differential responses to temperature variations were noted, with isolates SRA-1 and SRA-4 growing faster at different temperature regimes. Conidia count and biomass measurements revealed isolate-specific patterns under varying temperature regimes. Growth performance and colony characteristics varied among isolates. Environmental responses indicated sensitivity to osmotic potential and pH, as well as preferences for different culture media. This research provides crucial insights into the morphology, behavior, and adaptation of W. carpophilus. These findings will facilitate the establishment of management strategies against shot-hole disease in cherry orchards.