Agricultural grasses cover a major part of the land surface in temperate agro-ecosystems and contribute significantly to the formation of soil organic matter. Crop-derived lipids are assumed to be responsible for fast carbon turnover in soils. Differences in lipid distribution patterns between crops following C3 and C4 photosynthesis pathways have rarely been described, but could be useful for source apportionment of crop-derived input into soils or sediments. The distribution of long chain n-carboxylic acids (C22, C24, C26) reveals significant differences between crop plants following either the C3 or the C4 photosynthetic carbon fixation pathway. The plant compartments leaves, stems and roots of C4 plants contain relatively large proportions (> 40%) of n-C24 carboxylic acid when compared to C3 plants. These reveal larger relative proportions of n-C22 and n-C26 acids, whose relative abundance is subject to change between different plant compartments and during the growing season. The carboxylic acid ratio [CAR = n-C24/(n-C22 + n-C26) carboxylic acids] provides distinct ratios for C4 (> 0.67) and C3 crops (< 0.67) and can thus be used as a molecular marker for the differentiation of crop plant biomass. In combination with the bulk stable carbon isotopic composition (d13C) the CAR can be used as a tool for the estimation of the C4 derived carbon proportion in soils or sediments.