Our objectives were to investigate the concentration and composition of fatty acids (FA) and oxylipids (OxL) in the uterine fluid (UF) of lactating cows at early, mid, and late diestrus. After synchronization of the estrous cycle, cows (n = 30) were blocked by parity and assigned randomly to receive transcervical uterine flushing on d 5 (UFD5), 10 (UFD10) or 15 (UFD15) of the estrous cycle. Flushing was performed by infusion of 30 mL of PBS into the uterine horn ipsilateral to the corpus luteum. The recovered flushing was centrifuged and stored at −80°C. Samples in which the recovered volume was >15 mL without visible blood contamination (8 samples/group) were used for analyses of FA and OxL composition by LC-MS/MS. Internal standards were used to estimate the concentration of specific lipids. Data were analyzed by ANOVA using the PROC GLIMMIX of SAS. Statistical models included the effects of day, parity, and their interaction. A total of 29 FA and 63 OXL were identified. Concentration of total FA and total OxL tended (P < 0.10) to be greater in UFD15 (1.7 ng/mL and 62 pg/mL, respectively) than in UFD5 (0.9 ng/mL and 31 pg/mL) and UFD10 (0.8 ng/mL and 26 pg/mL). In addition, concentration of polyunsaturated FA (PUFA) and monounsaturated FA (MUFA) were greater (P < 0.05) in UFD15 than in UFD5 and UFD10. Concentration of SFA and the proportion of PUFA, MUFA, and SFA were similar between groups. Concentration of n-6 FA was greater (P < 0.05) and concentration of n-3 FA tended to be greater (P < 0.09) in UFD15 than in UFD5 and UFD10. The ratio n-6:n-3 did not differ between groups and averaged 3.7. Concentration of series-2 prostaglandins (S2PG), DGLA-OxL, and n-3-OxL were all greater (P < 0.05) in UFD15 than in UFD5 and UFD10. Proportionally, HODE OxL was reduced (P < 0.05) while S2PG, DGLA-OxL, and n-3-OxL were increased (P < 0.05) in UFD15 compared with UFD5, which were both similar to UFD10. In conclusion, multiple FA and OxL accumulate in the uterine lumen during diestrus, changing the abundance and profile of lipids in the UF. These changes are likely caused by rising concentrations of progesterone during diestrus and are relevant for early conceptus development and survival.