ABSTRACTRecently, a novel class of mechanically sensitive channels has been identified and have been called Piezo channels. In this study, we explored Piezo channel expression in sensory neurons supplying the guinea pig corneal epithelium, which have well‐defined modalities in this species. We hypothesized that a proportion of corneal afferent neurons express Piezo2, and that these neurons are neurochemically distinct from corneal polymodal nociceptors or cold‐sensing neurons. We used a combination of retrograde tracing to identify corneal afferent neurons and double label in situ hybridization and/or immunohistochemistry to determine their molecular and/or neurochemical profile. We found that Piezo2 expression occurs in ∼26% of trigeminal ganglion neurons and 30% of corneal afferent neurons. Piezo2 corneal afferent neurons are almost exclusively non‐calcitonin gene‐related peptide (CGRP)‐immunoreactive (‐IR), medium‐ to large‐sized neurons that are NF200‐IR, suggesting they are not corneal polymodal nociceptors. There was no coexpression of Piezo2 and transient receptor potential cation channel subfamily M member 8 (TRPM8) transcripts in any corneal afferent neurons, further suggesting that Piezo2 is not expressed in corneal cold‐sensing neurons. We also noted that TRPM8‐IR or CGRP‐IR corneal afferent neurons are almost entirely small and lack NF200‐IR. Piezo2 expression occurs in a neurochemically distinct subpopulation of corneal afferent neurons that are not polymodal nociceptors or cold‐sensing neurons, and is likely confined to a subpopulation of pure mechano‐nociceptors in the cornea. This provides the first evidence in an in vivo system that Piezo2 is a strong candidate for a channel that transduces noxious mechanical stimuli. J. Comp. Neurol. 522:2967–2979, 2014. © 2014 Wiley Periodicals, Inc.