PIWI-interacting RNAs (piRNAs) are ~25–33 nt small RNAs expressed in animal germ cells. Here, the authors develop a single-cell small RNA sequencing method and report that a class of ~20-nt piRNAs lacking 3′ end 2′-O-methylation are associated with PIWIL3 protein and predominantly expressed in human and monkey oocytes.