Potato is one of the most consumed crops worldwide. One strategy aimed at pathogen and pest management is the cultivation of resistant varieties. In this study, 352 genotypes from the Kazakhstan potato germplasm collection were screened for the presence of loci for resistance to the most harmful pathogens and pests using 30 DNA markers. ‘Rucheek’ and ‘Spiridon’, among the top global varieties, and ‘Janaisan’ and ‘Fedor’, among the top local varieties, were identified as bearing the most resistance loci in the collection, with at least 14 markers each. The specimens carrying the genes Ryadg (ADG1, ADG2), Nb (SPUD237), Ns (SC811, CP16), Rpi-phu1 (GP94), and GroV1 (X02), which demonstrate confirmed resistance to PVY, PVA, PVX, PVS, Phytophthora infestans, and Globodera rostochiensis, were most frequently found in the collection. Loci for resistance to Synchytrium endobioticum and Globodera pallida were less introgressed into the Kazakhstani cultivars and were almost absent in the germplasm collection. The less abundant loci found in the current potato collection were Ryadg (RysC3), Gro1-4 (Gro1-4), and Rladg (RGASC850). Screening of the potato collection showed that the markers SPUD237, CP60, 45/X1, and CT214 generated additional polymorphic amplicons, while markers Nl25, C237, GP179, and GP122718 were less applicable in robust large-scale screening. The results from this study may greatly contribute to marker-assisted selection and gene pyramiding strategies aimed at developing new potato varieties with multiple resistance to biotic stress