4.5SH RNA is a 94-nt small RNA with unknown function. This RNA is known to be present in the mouse, rat, and hamster cells; however, it is not found in human, rabbit, and chicken. In the mouse genome, the 4.5SH RNA gene is a part of a long (4.2 kb) tandem repeat ( approximately 800 copies) unit. Here, we found that 4.5SH RNA genes are present only in rodents of six families that comprise the Myodonta clade: Muridae, Cricetidae, Spalacidae, Rhizomyidae, Zapodidae, and Dipodidae. The analysis of complementary DNA derived from the rodents of these families showed general evolutionary conservation of 4.5SH RNA and some intraspecific heterogeneity of these RNA molecules. 4.5SH RNA genes in the Norway rat, mole rat, hamster and jerboa genomes are included in the repeated sequences. In the jerboa genome these repeats are 4.0-kb long and arranged tandemly, similar to the corresponding arrangements in the mouse and rat genomic DNA. Sequencing of the rat and jerboa DNA repeats containing 4.5SH RNA genes showed fast evolution of the gene-flanking sequences. The repeat sequences of the distantly related rodents (mouse and rat vs. jerboa) have no apparent similarity except for the 4.5SH RNA gene itself. Conservation of the 4.5SH RNA gene nucleotide sequence indicates that this RNA is likely to be under selection pressure and, thus, may have a function. The repeats from the different rodents have similar lengths and contain many simple short repeats. The data obtained suggest that long insertions, deletions, and simple sequence amplifications significantly contribute in the evolution of the repeats containing 4.5SH RNA genes. The 4.5SH RNA gene seems to have originated 50-85 MYA in a Myodonta ancestor from a copy of the B1 short interspersed element. The amplification of the gene with the flanking sequences could result from the supposed cellular requirement of the intensive synthesis of 4.5SH RNA. Further Myodonta evolution led to dramatic changes of the repeat sequences in every lineage with the conservation of the 4.5SH RNA genes only. This gene, like some other relatively recently originated genes, could be a useful model for studying generation and evolution of non-protein-coding genes.