Inferences regarding hybridization rely on genetic markers to differentiate parental taxa from one another. Intersimple sequence repeat (ISSR) markers are based on single-primer PCR reactions where the primer sequence is derived from di- and trinucleotide repeats. These markers have successfully been used to assay genetic variability among cultivated plants, but have not yet been tested in natural populations. We used genetic markers generated from eight ISSR primers to examine patterns of hybridization and purported examples of hybrid speciation in Penstemon (Scrophulariaceae) in a hybrid complex involving P. centranthifolius, P. grinnellii, P. spectabilis and P. clevelandii. This hybrid complex has previously been studied using three molecular data sets (allozymes, and restriction-site variation of nuclear rDNA and chloroplast DNA). These studies revealed patterns of introgression involving P. centranthifolius, but were unsuccessful in determining whether gene flow occurs among the other species, and support for hypotheses of diploid hybrid speciation was also lacking. In this study, we were able to fingerprint each DNA accession sampled with one to three ISSR primers and most accessions could be identified with a single primer. We found population- and species-specific markers for each taxon surveyed. Our results: (i) do not support the hybrid origin of P. spectabilis; (ii) do support the hypothesis that P. clevelandii is a diploid hybrid species derived from P. centranthifolius and P. spectabilis; and (iii) demonstrate that pollen-mediated gene flow via hummingbird vectors is prevalent in the hybrid complex.