Mass spectrometry-based untargeted lipidomics has revealed the lipidome atlas of living organisms at the molecular species level. Despite the double bond (C = C) position being a crucial factor in biological system, the C = C defined structures have not yet been characterized comprehensively. Here, we present an approach for C = C position-resolved untargeted lipidomics using a combination of oxygen attachment dissociation and computational mass spectrometry to increase the annotation rate. We validated the accuracy of our platform as per the authentic standards of 85 lipids and the biogenic standards of 52 molecules containing polyunsaturated fatty acids (PUFAs) from the cultured cells fed with various fatty acid-enriched media. By analyzing human and mice-derived samples, we characterized 648 unique lipids with the C = C position-resolved level encompassing 24 lipid subclasses defined by LIPIDMAPS. Our platform also illuminated the unique profiles of tissue-specific lipids containing n-3 and/or n-6 very long-chain PUFAs (carbon ≥ 28 and double bonds ≥ 4) in the eye, testis, and brain of the mouse. Mass spectrometry is a powerful approach for untargeted lipidomics, however, the unambiguous determination of double bond positions remains challenging. Here, the authors present an approach for double bond position-resolved untargeted lipidomics using a combination of oxygen attachment dissociation and computational mass spectrometry to accurately annotate the biologically relevant lipidome. [ABSTRACT FROM AUTHOR]