Concern over potential exposures of ionizing radiation (IR) to large populations has emphasized the need for rapid and reliable methods of biodosimetry to determine absorbed dose and required triage. Lipidomics has emerged as a powerful technique for large-scale lipid identification and quantification. Indirect effects from IR exposure generate reactive oxygen species (ROS) through water hydrolysis and may subsequently damage cellular lipids. Thus, rapid identification of specific affected lipid molecules represents possible targets for biodosimetry. The current study addresses temporal changes in the serum lipidome from 4 h to 28 d in nonhuman primates (NHPs) with radiation-induced hematopoietic syndrome (6.5 Gy exposure, LD50/30). Statistical analyses revealed a highly dynamic temporal response in the serum lipidome after IR exposure. Marked lipidomic perturbations occurred within 24 h post-irradiation along with increases in cytokine levels and C-reactive protein. Decreases were observed in di- and triacylglycerides, sphingomyelins (SMs), lysophosphatidylcholines (LysoPCs), and esterified sterols. Conversely, free fatty acids and monoacylglycerides significantly increased. Decreased levels of SMs and increased levels of LysoPCs may be important markers for biodosimetry ~2 d-3 d post-irradiation. The biphasic and dynamic response to the serum lipidome post-irradiation emphasize the importance of determining the temporal long-term response of possible radiation markers.