Macrolophus pygmaeus (Rambur, 1839) (Insecta, Heteroptera, Miridae) is a predator of key vegetable crop pests applied as a biocontrol agent in the Mediterranean region. M. pygmaeus and M. melanotoma (A. Costa, 1853) are cryptic species with great morphological similarity which results in their misidentification and negative consequences for the conservation of their populations on greenhouse and outdoor crops. In order to find out specific markers for their separation we studied the karyotype, male meiosis and heterochromatin composition of these species and additionally of a third species (as a reference one), M. costalis Fieber, 1858. We demonstrate here that all the three species share achiasmate male meiosis and sex chromosome pre-reduction. On the other hand, the species differ in karyotype, with 2n=28 (26+XY) in M. pygmaeus, 2n=27 (24+X1X2Y) in M. costalis, and 2n=34 (32+XY) in M. melanotoma, and heterochromatin distribution and composition. In addition, the species differ in sperm morphology: sperm cells of M. costalis are significantly longer with longer head and tail than those of M. melanotoma and M. pygmaeus, whereas sperm cells of M. melanotoma have a longer tail than those of M. pygmaeus. All these characters can be used as markers to identify the species, in particular the cryptic species M. melanotoma and M. pygmaeus.