Low-molecular weight RNA (LMW RNA) analysis using staircase electrophoresis was performed for several species of eukaryotic and prokaryotic microorganisms. According to our results, the LMW RNA profiles of archaea and bacteria contain three zones: 5S RNA, class 1 tRNA and class 2 tRNA. In fungi an additional band is included in the LMW RNA profiles, which correspond to the 5.8S RNA. In archaea and bacteria we found that the 5S rRNA zone is characteristic for each genus and the tRNA profile is characteristic for each species. In eukaryotes the combined 5.8S and 5S rRNA zones are characteristic for each genus and, as in prokaryotes, tRNA profiles are characteristic for each species. Therefore, stable low molecular weight RNA, separated by staircase electrophoresis, can be considered a molecular signature for both prokaryotic and eukaryotic microorganisms. Analysis of the data obtained and construction of the corresponding dendrograms afforded relationships between genera and species; these were essentially the same as those obtained with 16S rRNA sequencing (in prokaryotes) and 18S rRNA sequencing (in eukaryotes).