Duck hepatitis A virus genotype 3 (DHAV-3) is the most popular pathogen of duck viral hepatitis (DVH) and has led to a huge economic threat to the Asian duck industry. In this work, we investigated the differences in the LC-MS/MS-based dynamic lipid profiles between susceptible and resistant Pekin duck lines with DHAV-3 infection. We found that the plasma lipidome of the two duck lines was characterized differently in expression levels of lipids during the infection, such as decreased levels of glycerolipids and increased levels of cholesteryl esters and glycerophospholipids in susceptible ducks compared with resistant ducks. By integrating lipidomics and transcriptomics analysis, we showed that the altered homeostasis of lipids was potentially regulated by a variety of differentially expressed genes including CHPT1, PI4K2A, and OSBP2 between the two duck lines, which could account for liver dysfunction, apoptosis, and illness upon DHAV-3 infection. Using the least absolute shrinkage and selection operator (LASSO) approach, we determined a total of 25 infection-related lipids that were able to distinguish between the infection states of susceptible and resistant ducks. This study provides molecular clues for elucidating the pathogenesis and therapeutic strategies of DHAV-3 infection in ducklings, which has implication for the development of resistance breeding.