Heat stress (HS) is a global challenge for the dairy industry; however, its abnormal physiological alterations still remain elusive. Here, a LC-MS-based study on lysophosphatidylcholine (lysoPC) and phosphatidylcholine (PC) in plasma of HS dairy cows was performed to explore HS-induced metabolic alterations. LysoPCs or PCs exist in 2 forms/isomers, with the fatty acyl groups at positions 1 (sn-1) or 2 (sn-2) on the glycerol backbone. First experiment, the LC-MS-based method of discriminating isomers of lysoPCs or PCs was constructed. The isomeric lysoPCs or PCs can be differentiated by chromatographic separations and relative intensities of their characteristic fragments. Second experiment, the enrolled 2 groups of Holstein cows were in second parity, mid-lactation stage, had similar milk yield (both around 40 kg/day), and fed the same diet. The HS-free group consisted of 22 cows, with plasma samples obtained in spring season, after temperature-humidity index (THI) was around 50–55 for 1 mo. The HS group consisted of another 22 cows, with samples obtained in summer season, after THI gradually increased from 68 to 80 over 1 mo. LC-MS in combination with multivariate analyses on plasma profiling of HS-free and HS groups was conducted to discover HS-induced discriminating metabolites (P < 0.05), from which isomeric lysoPCs or PCs were identified by the constructed method in first experiment. The results revealed that concentrations of 14 lysoPCs and 17 PCs were significantly up- or downregulated in HS compared with in HS-free group, indicating HS-induced alterations of lysoPCs and PCs. Of these 31 discriminating metabolites, concentrations of sn-1 lysoPC 14:0, sn-2 lysoPC 15:0, sn-1 lysoPC 18:0, and sn-2 lysoPC 22:4 were upregulated in HS group (P < 0.05), concentrations of sn-1 PC 14:0/18:3, sn-1 PC 14:0/18:2, sn-2 PC 15:0/17:1, sn-1 PC 14:0/20:4, sn-1 PC 18:0/15:1, sn-1 PC 18:0/15:0, and sn-2 PC 15:0/20:4, were downregulated in HS group (P < 0.05); whereas, concentrations of their respective isomers remained unchanged, suggesting isomers of these lysoPCs and PCs have different biological functions played in lactating dairy cows under HS states.