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Numerous studies have indicated that yellow semen syndrome (YSS) of turkey is associated with the production of low semen quality, resulting in reduced fertility and hatchability. It is unknown at present if the etiology of YSS also could be linked to low-molecular weight metabolites. The aim of this study was to examine the metabolome of white and yellow seminal plasma of turkeys. Two different metabolomics approaches, shotgun (direct infusion) and liquid chromatography-mass spectrometry (LC-MS), were employed to identify metabolites differentially abundant in yellow seminal plasma. Significant changes in the levels of 1549 and 2093 metabolites were detected in yellow vs. white seminal plasma using shotgun and LC-MS, respectively. Of these, 354 metabolites (189 increased and 165 decreased) after shotgun and 936 metabolites (363 increased and 573 decreased) after LC-MS were putatively identified using the Human Metabolome Database. Significantly differentiated metabolites were subjected to Ingenuity Pathway Analysis. Lipid metabolism, molecular transport, and nucleic acid metabolism were the top pathways that differentiated white and yellow seminal plasma. These data strongly suggest that disturbance of carbohydrate and lipid metabolism is characteristic for YSS. The abnormal metabolism of lipids may contribute to the numerous lipid vacuoles previously observed in the reproductive tracts of YSS males. An increased level of riboflavin in YSS may be responsible for yellow turkey semen pigmentation. A disturbance in thyroid hormone metabolism visible at protein and metabolic levels may be involved in YSS in turkey. The low quality of YSS may be linked with the presence of drug residues in the reproductive tract.