The present study was aimed to evaluate the genetic diversity and pattern of variation throughout the ranges of polymorphic aggregate group C. biflorus in Bulgaria. For this purpose, samples from five Crocus L. species collected from 15 natural populations were assessed using ISSR molecular markers. Cluster dendrogram was created using the Wars’s method (PAST 4.03), then combined into a consensus tree using Consense (PHYLIP 3.69). Our results showed significant levels of genetic variation of the C. biflorus group corresponding with morphological diversity. Dendrogram showed that the samples of C. adamioides were in the group of C. biflorus but cannot be grouped with C. biflorus subsp. adamii. The previous analysis based on ITS sequences confirms that the population belongs to C. adamioides. The obtained data confirmed that C. chrysanthus was a separate taxon. The results also verified the genetic divergence of the C. cf. biflorus. The ISSR analysis showed the need for additional DNA-based methods for taxonomic revisions of the genus Crocus L.