Feline Panleukopenia Virus (FPV) is a highly contagious and systemic virus that is environmentally stable, targets leukocytes, and affects cats of all ages. Within the United States, particularly in the population-dense downstate region of New York, no molecular surveillance or strain isolation has been conducted for FPV. The main goal of this study is to conduct molecular and serological surveillance of FPV among some shelter cats in this area and to do isolation and genome mining of some of the detected isolates. We used samples (swabs and sera) from 109 shelter cats by qPCR and immunofluorescent assay (IFA) respectively. Our results show that 25.0% (29/108) of the cats tested were FPV positive by qPCR, and 39.4% (43/109) of the cats tested FPV positive by IFA. FPV Viral isolation and identification were conducted using Madin-Darby Canine Kidney (MDCK) cells. Next Generation Sequencing (NGS) confirmed the presence of a novel FPV isolate (Accession: PZ251627) circulating within the tested shelter cat population. Phylogenetic analysis showed that the sequence of the reported FPV isolate had the highest full-length sequence similarity with isolates MH165482 at 99.3%, MN127781 at 99.2%, and MH165481 at 99.2%. Additionally, phylogenetic analysis of the VP2 genome sequence showed the following highest sequence similarities; OQ615264 at 99.2%, KT899746 at 99.1%, and PVMRFD at 99.3%. The following substitutions were noted in comparison of the isolate to the reference sequence (MN45165): Ile101Thr and Glu411Ala. Inverse distance weighting (IDW) interpolation indicated the presence of a higher occurrence of FPV-positive cats in the western region of Downstate New York, encompassing the areas of the 5 boroughs of NYC and Nassau County. Continued FPV surveillance in cats in this region is highly recommended.