Reconstructions of historical primary production, and of the algal groups and habitats that contribute to it, are fundamental in studies of climate and environmental change in both marine and freshwater environments. The aims of this study were to critically evaluate morphological and biogeochemical markers of diatom production by direct comparison of diatom marker pigments with absolute diatom biovolume and to partition diatom production between the main habitats (plankton, sea ice, and benthos). Sediments in two cores from the Larsemann Hills, Antarctica, spanning the last 10,000 yr, were analyzed for siliceous microfossils by microscopy and for fossil pigments by high-performance liquid chromatography. Diatom pigments (diadinoxanthin, diatoxanthin, fucoxanthin) were highly correlated (r(2) = 0.557 and 0.358, p < 0.0001) with diatom biovolume in the marine intervals of both cores, but only weakly correlated in the lacustrine sections (r(2) = 0.102, p = 0.111; r(2) = 0.223, p = 0.001, after correction for temporal autocorrelation), possibly because of frustule dissolution and selective degradation of diadinoxanthin and diatoxanthin. In contrast, fucoxanthin was better preserved. By combining both microfossil and pigment proxies, we obtained a first estimate of diatom production in specific habitats (benthic and planktonic). Benthic diatom production was greatest in the lacustrine core sections, when benthic microbial mats dominated the flora, whereas diatoms were associated mainly with the water column and sea ice during the marine intervals. The combination of both proxies in marine and freshwater environments permits more accurate interpretation of pigment and diatom data in paleo- and neoecological research and the partitioning of diatom production between habitats.