Using a novel small RNA cloning method, we identified 630 piRNA-like RNAs (pilRNAs) from the mouse testis, and 498 of them are novel. These pilRNA genes were mapped to all chromosomes as 71 clusters, and the majority of them ( approximately 84%) are derived from intergenic, intronic, and exonic sequences. One of the structural characteristics for pilRNAs is that a single locus can encode numerous homologous pilRNAs with overlapping sequences. Hundreds or even thousands of pilRNAs from a single pilRNA gene cluster are all produced from a single long transcript. Expression profiling for 64 pilRNAs revealed that approximately 14% of all the pilRNAs analyzed displayed a ubiquitous expression pattern, although the majority of ( approximately 86%) pilRNAs were preferentially or exclusively expressed in meiotic and haploid male germ cells of the testis. Our semiquantitative analyses also suggest that the testis is the organ with the highest expression of pilRNAs both in number and in abundance. The large number, high abundance, unique genomic locations, and biogenesis all suggest that pilRNAs have important regulatory roles not only in spermatogenesis but also in other biological processes.