Macrobiotus polonicus and Macrobiotus persimilis are two species of the “Macrobiotus hufelandi group” which are distinguished one from the other for the presence in M. polonicus of one lateral gibbosity on each hind leg, the size of cuticular pores (smaller and less evident in M. polonicus), the buccal tube size (larger in M. persimilis) and the egg processes (larger terminal discs and with more evident indentation in M. polonicus). However, the two taxa share peculiar characteristics: similar values of the pt index relative to the insertion point of the stylet supports on the buccal tube, first macroplacoid clearly longer than the second and with a central constriction, lunules of the hind legs clearly larger than those of the first three pairs and with an indented margin, egg shell smooth or with faint dots but without a reticular design. Therefore, it could be hypothesized that they belong to a single peculiar group, namely the “Macrobiotus polonicus/persimilis group”. In order to verify this hypothesis, a morphological and DNA barcoding analysis (cox1) was carried out on tardigrades attributable to these two taxa and coming from five different localities in Italy and France. This study revealed differences, both morphological and molecular, between some populations apparently belonging to the same species, which in some cases turned out to be cryptic species. A phylogenetic analysis using rDNA 18S was performed in order to verify the relationships among the species of this group and more in general among those of the “M. hufelandi group”. The DNA barcoding analysis revealed a high divergence, with very high values of genetic distance among some populations (more than 18%). In one case (Enna, Sicily) at least two different and very distinct entities are present together. The morphological and morphometrical investigation, also based on type material, confirms this variety, showing that at least four species related to M. polonicus and three related to M. persimilis should be distinct. In several cases the relationship between morphology and cox1 sequence has been ascertained. The dendrogram computed on 18S showed that there are two different sequences pertaining to the “M. polonicus/persimilis group” clustered in a single evolutionary line inside the main M. hufelandi cluster.