Interspecific hybridization can result in significant shifts in allele frequencies. The objective of the present study was to assess the level of genetic variation in populations of P. mariana × P. rubens hybrids derived from artificial crosses. Progenies from backcross populations created through a series of controlled pollinations among P. mariana and P. rubens trees across the hybridization index were analyzed. Several Inter Simple Sequence Repeat (ISSR) and Random Amplified Polymorphic DNA (RAPD) primers were used to amplify genomic DNA samples from each population. ISSR primers produced from 30% to 52% polymorphic loci. The level of polymorphism was higher with RAPD mark- ers, ranging from 57% to 76%. Overall, the two marker systems generated similar levels of polymorphic loci for P. mariana and P. rubens populations. No significant differences were found among the P. mariana × P. rubens popula- tions analyzed and between the hybrids and the parental populations regardless of the molecular marker used. This con- firms the genetic closeness of P. mariana and P. rubens species.