As far as an unisexuality in the animal kingdom is typical, in higher plants there most species are bisexual. Whereas in haploid bryophyte it can be noticed that dioecy in this group prevails. In the dioecious bryophyte a huge problem makes diagnostics of gender. Most dioecious mosses do not exhibit any discernible sexual dimorphism. The aim of present research was a finding genetic differences between two gender in the dioecious moss Nyholmiella obtusifolia with use of ISSR markers. This study was conducted on 16 individuals: 6 females (without sporophytes) and 10 randomly selected (unknown gender), what was dedicated by a very small number of sex-expressing plant material (rare sporophytes). Out of 12 ISSR primes using to this research, only 4 of them gave clear band patterns. There were: IS807, IS825, IS835 and IS840. The following results are based only on the amplification with ISSR 807 primer. It revealed the products of amplification, which seem to be typical for males and females, which sizes were approximately 600 bp for the former and 500 bp for the latter. This study without doubt is the first trial of finding of genetic differences between females and males in dioecious moss Nyholmiella obtusifolia. This is the first step in development of sex-specific molecular marker for Nyholmiella obtusifolia. It is insomuch important, because it would be just the second moss, for which a molecular sex marker would be devised.