In plants there are several classes of 21–24-nt short RNAs that regulate gene expression. The most conserved class is the microRNAs (miRNAs), although some miRNAs are found only in specific species. We used high-throughput pyrosequencing to identify conserved and nonconserved miRNAs and other short RNAs in tomato fruit and leaf. Several conserved miRNAs showed tissue-specific expression, which, combined with target gene validation results, suggests that miRNAs may play a role in fleshy fruit development. We also identified four new nonconserved miRNAs. One of the validated targets of a novel miRNA is a member of the CTR family involved in fruit ripening. However, 62 predicted targets showing near perfect complementarity to potential new miRNAs did not validate experimentally. This suggests that target prediction of plant short RNAs could have a high false-positive rate and must therefore be validated experimentally. We also found short RNAs from a Solanaceae-specific foldback transposon, which showed a miRNA/miRNA*-like distribution, suggesting that this element may function as a miRNA gene progenitor. The other Solanaceae-specific class of short RNA was derived from an endogenous pararetrovirus sequence inserted into the tomato chromosomes. This study opens a new avenue in the field of fleshy fruit biology by raising the possibility that fruit development and ripening may be under miRNA regulation.