Bulgaria was the secondary gene pool for many crops, and one of the first was pepper. However, during the political transforming and economic crises, the lands for growing pepper (Capsicum spp.) were reduced, and thereafter, the genetic diversity was lost. With pepper, Bulgaria still has priority providing on European scale valuable pepper germplasm, and this priority should be evaluated and preserved. We present our efforts to characterize pepper accessions using RAPD as well as the retroelement-based Inter-SINE Amplified Polymorphism (ISAP) method initially developed for potatoes. Several short interspersed nuclear element (SINE) families were active within the common ancestor of potato and pepper. We studied the degree of polymorphisms in a collection of 73 pepper genotypes, divided into six groups, using ISAP with primers derived from seven Solanaceae SINE families as well as two subfamilies. Two primer pairs from the families SolS-II and SolS-V generated the most fragments and most informative banding patterns. These SINE-based ISAP reactions are best suited for identifying species of the Capsicum genus. The most polymorphic profiles within all studied were generated by C. baccatum accessions. In contrast, intraspecific application of the SINE-based markers yielded a high percentage of conserved ISAP fragments. From a total of 56 C. annuum accessions, only three of them with two different profiles were identified. Our results demonstrate that potato-based SolS-SINE primers can be adapted for molecular genotyping in peppers. The low intraspecies polymorphism generated by ISAP forced us to investigate RAPD as an alternative low-cost genotyping approach. RAPD was successfully applied on a group of mutant lines and corresponding source lines, carrying valuable breeding traits. Despite the low polymorphic levels, we have identified four RAPD primers, capable to discriminate among several genotypes.