Fungi of the genus Colletotrichum are causal agents of plant diseases with constantly growing economic importance. Accurate pathogen identification is a significant prerequisite for effective disease control. The aim of the present investigation was to clarify the species affiliation of Colletotrichum isolates obtained from different hosts in Bulgaria and to determine the phylogenetic relationships between them by applying DNA barcoding. Thirty-five fungal isolates obtained from five botanical families (Solanaceae, Rosaceae, Musaceae, Cucurbitaceae and Caryophyllaceae) were morphologically characterized and subjected to molecular analysis based on four fungal barcode markers – the primary ITS barcode and the secondary marker regions ACT, EF-1a and TUB2 (Fig. 1). Three of the barcodes (ITS, ACT and TUB2) showed complete success rate of PCR amplification and sequencing and proved efficient for reliable identification at species level. BLAST analyses identified eleven Colletotrichum species assigned to five different complexes – C. coccodes, C. acutatum, C. gloeosporioides, C. dematium and C. spaethianum. The resolution power of ITS region was not sufficient to discriminate interspecies variations within C. coccodes, C. dematium and C. spaethianum complexes confirming the requirement for secondary barcodes in order to resolve the genetic variability of the Colletotrichum isolates. DNA barcoding analyses revealed that the highest species variation was observed among the isolates from pepper (Capsicum annuum). Interestingly, an isolate from the same host identified as C. truncatum on the basis of morphological characters appeared to be C. circinans when applying DNA barcode markers. According to our knowledge, this species has not been reported as a causal agent of pepper anthracnose. Data obtained in this study improve our understanding of the genetic diversity within the Colletotrichum population pathogenic on cultivated plants in Bulgaria.