Heat stress (HS) by high-temperature environment reduced the production performance of poultry and caused losses to the breeding industry. The present study was conducted to investigate the effects of HS on serum lipidomics in Chinese indigenous slow-growing broiler chickens (Huaixiang chickens). A total of 40 8-week-old female Huaixiang chickens were randomly allocated to two groups, including normal temperature (NT, fed basal diet) and HS (fed basal diet), and each group consisted of five replicates with four birds per replicate. NT and HS groups were exposed to 21.3 ± 1.2 °C and 32.5 ± 1.4 °C for 4 weeks, respectively. Serum lipidomics in broilers was determined by liquid chromatography-mass spectrometry (LC-MS)-based metabolomics. The results indicated that there were significant differences in metabolic spectra between the groups, and a total of 17 differential metabolites were screened. Compared with NT group, HS group reduced the serum ceramide (cer) (d18:1/22:0), cer (d18:1/24:1), cer (d20:2/22:2), lyso-phosphatidylcholine (LPC) (18:0), phosphatidylcholine (PC) (18:0/20:4), PC (15:0/23:4), PC (18:0/22:6), PC (18:2/18:2), phosphatidylethanolamine (PE) (18:1/18:1), polyethylene terephthalate (PEt) (37:3/8:0), phosphatidylglycerol (PG) (32:1/16:2), phosphatidyl methyl ethanolamine (PMe) (19:3/13:0), PMe (26:1/9:0), sphingomyelin (SM) (d16:0/18:1), triglycerides (TG) (18:0/18:1/18:2), and TG (19:4/21:6/21:6) levels [variable importance in the projection (VIP > 1 and P < 0.05)], while HS group increased serum PC (17:0/17:0) content (VIP > 1 and P < 0.05). Also, metabolic pathway analysis showed that the pathways of glycerolphospholipid, linoleic acid and α-linolenic acid metabolism, and glycosylphosphatidylinositol (GPI)-anchored biosynthesis were changed (P < 0.05). In conclusion, HS led to the disorders of serum lipid metabolism in broilers, and mainly downregulated serum content of phospholipids. These findings provide novel insights into the effects of HS on serum lipidomics in indigenous slow-growing chickens.