In this study, we aimed to identify metabolite signatures that characterize metritis prior to, during, and after the disease incidence. Blood samples were collected from 100 Holstein cows at five time points before and after parturition. Six cows that developed metritis and 20 controls were selected for metabolomics analysis in a nested case-control study. Twenty nine serum metabolites were quantified using gas chromatography–mass spectroscopy. Results showed that similar panels of metabolites differentiated pre-metritic and control cows at 8 and 4 wk prepartum. The top most important metabolites that differentiated the two groups of cows at 8 wk prepartum were oxalate, ornithine, pyroglutamic acid, d-mannose, and glutamic acid, and at 4 wk prepartum were ornithine, pyroglutamic acid, d-mannose, glutamic acid, and phosphoric acid, suggesting their potential use as risk biomarkers for metritis. Area under the curve with values of 1.0 and 0.969 at 8 and 4 wk, respectively, indicated that those panels of metabolites have a very high sensitivity and specificity to be used as risk biomarkers for metritis. Overall, results showed that specific serum metabolite signatures can be used to screen cows for susceptibility to metritis during the dry off period, and to better understand the etiopathobiology of the disease.