Piwi-interacting RNAs (piRNAs), a class of 26- to 32-nt non-coding RNAs (ncRNAs), function in germline development, transposon silencing, and epigenetic regulation. We performed deep sequencing and annotation of untreated and periodate-treated small RNA cDNA libraries from human fetal and adult germline and reference somatic tissues. This revealed abundant piRNAs originating from 150 piRNA-encoding genes, including some exhibiting gender-specific expression, in fetal ovary and adult testis—developmental periods coinciding with mitotic cell divisions expanding fetal germ cells prior to meiotic divisions. The absence of reads mapping uniquely to annotated piRNA genes demonstrated their paucity in fetal testis and adult ovary and absence in somatic tissues. We curated human piRNA-expressing regions and defined their precise borders and observed piRNA-guided cleavage of transcripts antisense to some piRNA-producing genes. This study provides insights into sex-specific mammalian piRNA expression and function and serves as a reference for human piRNA analysis and annotation. Display omitted •150 piRNA genes were identified in human adult testis and fetal ovary•Adult testis and fetal ovary differentially express piRNA genes•piRNAs were not identified in non-germline tissue•Non-repetitive piRNA genes account for over 90% of piRNAs Williams et al. identify and characterize 150 piRNA genes in the adult human testis and fetal ovary. Expression of piRNA genes differs between the sexes, and repetitive elements, including those for retrotransposons, are underrepresented among piRNA genes. No piRNA expression was detected in somatic tissue.