Maternal perceived prenatal stress (PPS) is a known risk factor for diverse developmental impairments in newborns, but the underlying molecular processes are incompletely understood. Here, we report that maternal PPS altered the birth profiles of blood transfer RNA fragments (tRFs), 16–50 nt long non-random cleavage products of tRNAs, in a sex-dependent manner. Importantly, comparing stressed versus control maternal and umbilical cord blood serum presented alterations that were not limited to individual tRFs, but rather reflected selective changes in particular tRF families grouped by their mitochondrial or nuclear genome origin, parental tRNA coded amino acid, and cleavage type. Specifically, tRF families that show stress- and sex-specific effects, revealed shared length and expression patterns which were strongest in the female newborns. Several of these tRFs carry complementary motifs to particular cholinergic mRNAs, suggesting possible translational regulation similar to microRNAs. Compatible with the cholinergic regulation of stress reactions, those “CholinotRFs” achieved an AUC of 95% when classifying female newborns according to maternal PPS. Moreover, we found altered catalytic activity of serum acetylcholinesterase, which was particularly elevated in male newborns, marking a second sex-specific effect. Our findings demonstrate an association of tRF families’ patterns with newborns’ sex-specific stress response to PPS and may lead to better diagnosis and therapeutic tools for these and other stressors.