We aim to evaluate the feasibility of Raman spectroscopy for parathyroid gland (PG) identification during thyroidectomy. Using a novel side-viewing handheld Raman probe, a total of 324 Raman spectra of four tissue types (i.e., thyroid, lymph node, PG, and lipid) commonly encountered during thyroidectomy were rapidly (< 3 s) acquired from 80 tissue sites (thyroid [n = 10], lymph node [n = 10], PG [n = 40], lipid [n = 20]) of 10 euthanized Wistar rats. Two partial least-squares (PLS)-discriminant analysis (DA) detection models were developed, differentiating the lipid and nonlipid (i.e., thyroid, lymph node, and PG) tissues with an accuracy of 100%, and PG, lymph node, and thyroid could be detected with an accuracy of 98.4%, 93.9%, and 95.4% respectively. This work demonstrates the feasibility of Raman spectroscopy technique for PG identification and protection during thyroidectomy at the molecular level.