Phospholipids such as phosphatidylcholines (PC), phosphatidylethanolamines (PE), phosphatidylinositols (PI), and phosphatidylserines (PS), have been proposed to be involved with compromised liver function in dairy cows. Examining changes in individual phospholipids may identify potential therapeutic targets of diseases in early lactation. We employed an untargeted mass spectrometry-based lipid profiling strategy to analyze simultaneously individual phospholipids in serum. The objective of this study was to examine whether individual phospholipids in serum may serve as disease risk indicators of mastitis, retained placenta, or both. 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 phospholipids were measured. Using PROC MIXED, serum levels of phospholipid classes and subclasses were compared between groups. Total PC, lysoPC, and lysoPE levels decreased until calving (all P < 0.0001). At calving, cows that subsequently developed mastitis had lower lysoPC, lysoPE, and inositol phosphate levels than cows that subsequently developed RP or remained healthy (lyso PC: both P = 0.03; lyso PE: both P = 0.02; inositol phosphate: P = 0.04 and P = 0.03) and thus, could be used as MA indicators. In contrast, RP cows had lower levels of highly unsaturated PS (PS with 4 double bonds) than MA or Healthy cows (both P = 0.02), which could serve as RP indicator. These findings suggest changes in phospholipids are an early indicator for the development of retained placenta and mastitis in multiparous cows and may be potential therapeutic targets for diseases in early lactation.