Argonaute/Piwi proteins associate with small RNAs that typically provide sequence specificity for RNP function in gene and genome regulation. Here we show that Twi12, a Tetrahymena Piwi protein essential for growth, is loaded with mature tRNA fragments. The tightly bound ~18-22 nt tRNA 3’ fragments are biochemically distinct from the tRNA halves produced transiently in response to stress. Notably, the end positions of Twi12-bound tRNA 3' fragments precisely match RNAs detected in total small RNA of mouse embryonic stem cells and human cancer cells. Our studies demonstrate unanticipated evolutionary conservation of mature tRNA processing to tRNA-fragment small RNAs. Two libraries are analyzed here: sRNAs associated with slightly overexpressed ZZ-tagged Twi12 purified under native conditions (size range 15-34nt), and those associated after formaldehyde crosslinking (15-22nt).