The need for multi-gene analysis in evolutionary and taxonomic studies is generally accepted. However, sequencing of multiple genes is not always possible. For various reasons, short mito-chondrial DNA barcodes are the only source of molecular information for some species in many genera, although multilocus data are available for other species of the same genera. In particular, such a situation exists in the species-rich butterfly subgenus Polyommatus (Agrodiaetus). Here, we analysed partitioning of this subgenus into species groups by using three data sets. The first data set was represented by short mitochondrial DNA barcodes for all analysed samples. The second and third data sets were represented by combination of short mitochondrial DNA barcodes for a part of the taxa with longer mitochondrial sequences COI+tRNA-Leu+COII (data set 2) and with longer mitochondrial COI+tRNA-Leu+COII and nuclear 5.8S rDNA+ITS2+28S rDNA sequences (data set 3) for the remaining species. We show that DNA barcoding approach (data set 1) failed to reveal the taxonomic structure resulting in numerous polytomies in the phylogenetic tree ob-tained. Combined analysis of the mitochondrial and nuclear sequences (data sets 2 and 3) re-vealed the species groups and position within these species groups even for the taxa for which only short DNA barcodes were available.