The study aimed to determine the genetic variability in pitch pine (Pinus rigida Mill.) growing in the Niepołomice Forest (southern Poland). In the late nineteenth and early twentieth century, Adolf Cieślar of the Department of Forestry Research in Mariabrunn near Vienna, Austria established the experimental crops of pitch pine. During the study, 227 trees that grew in seven subunits were considered; an analysis of genetic polymorphism using the intersimple sequence repeats (ISSR) technique revealed that pitch pine is genetically variable. The average number of alleles at a given locus for all the pine trees was 1.649, while the effective number of alleles at the loci was 1.435. The value of expected heterozygosity was 0.254, while the percentage of polymorphic loci was 75.30%. The average genetic distance between the examined pines was 0.082. Principal coordinate analysis (PCoA) divided the examined pines into three groups, which was also confirmed by the structure-analysis results of the software STRUCTURE. The resulting division was mainly generated by the SR70 primer, which was indicated to be the primer that differentiated the examined populations of pitch pine. Affiliation of particular trees to selected groups was based on their occurrence in individual crops. This suggests a different origin of the seeds used to establish the research plots of pitch pine in the Niepołomice Forest.