We previously documented that elevated serum nonesterified fatty acids (NEFA) before calving are a general disease risk indicator in dairy cows. Examining changes in individual or subclasses of NEFA may improve disease risk detection. Untargeted lipid profiling using ultra performance liquid chromatography-high resolution mass spectrometry (UPLC-MS) is a comprehensive technique that allowed simultaneous analysis of individual NEFA in complex biological samples. The objective of this study was to examine the association between serum levels of individual or subclasses of NEFA and subsequent development of diseases. Serum samples were collected from 161 multiparous cows 3, 2, and 1 week before calving and at calving. For this nested case-control study, serum samples of 3 groups of cows that either developed after calving retained placenta (RP: n = 8), mastitis (MA: n = 8), or remained healthy (Healthy: n = 9) were selected and serum levels of individual NEFA were measured. Using PROC MIXED, serum NEFA levels were overall and individually compared between groups. Levels of 22 individual NEFA between C14:0 and C26:0 were above the limit of detection. Overall levels of NEFA identified by UPLC-MS analysis were in good agreement with chemical analysis of total NEFA concentrations. Cows that developed RP and MA in early lactation had 1 week before calving (P = 0.05) and at parturition (P = 0.005) elevated overall NEFA levels compared with Healthy cows. Similar directional changes were observed for C14:0, C16:0, C16:1, C18:1, as well as the C18:1 to C18:0 ratio, which could serve as general disease risk indicators. In contrast, cows that subsequently developed diseases had lower elongation and/or very long chain fatty acid (≥C20) desaturation indices compared with Healthy cows. MA cows had greater ≥ C20 n6 to ≥ C20 n3 and C20:1 to C20:0 ratios and lower ≥ C20:0 levels compared with Healthy and RP cows, which could be used as potential MA indicators. These findings suggest changes in very long chain fatty acids levels and their ratios are an early indicator for the development of retained placenta and mastitis in multiparous cows.