The lipid content of sea ice samples collected in 2011 and 2012 from Resolute Passage in the Canadian Arctic Archipelago was measured and compared with related samples obtained from the Amundsen Gulf in 2008. The highly branched isoprenoid (HBI) sea ice biomarker, IP25, was found in sea ice samples from each study, consistent with its formation by diatoms during the spring bloom. Our analysis also revealed the occurrence of a number of sterols in Arctic sea ice and these were rigorously identified and quantified for the first time. Concentrations of IP25 and sterols exhibited some variability between sampling studies, with somewhat higher values in samples from Resolute in 2012 than for the other two datasets, consistent with a general increase in biomass; however, major differences in biomarker concentration between sampling studies were not observed. An estimate of the proportion of Arctic sea ice diatoms that produce IP25 (ca. 1–5%) was obtained by comparison of the concentration of IP25 in the samples with those in laboratory cultures of known HBI-producing diatoms and cell enumeration in selected sea ice samples. The estimate is similar to the proportion of Haslea spp. in the same samples, providing further support to the suggestion that at least some species of the Haslea genus may be responsible for the biosynthesis of IP25 and related HBI diatom lipids in Arctic sea ice and that IP25 is made by a relatively small proportion of sea ice diatoms. In contrast, median sterol/IP25 values were all substantially higher than those in cultures of HBI-producing diatoms, suggesting that sterols are made by the majority of sea ice diatoms. The sterol/IP25 ratio was quite variable between locations and samples, likely as a result of differences in diatom assemblage; however, a comparison of individual and median sterol/IP25 values in sea ice with those from surface sediments from different Arctic regions demonstrated that sterols from sea ice diatoms may, in some cases, have a significant impact on the sedimentary budget. This should be considered carefully for quantitative estimates of palaeo sea ice reconstruction using methods such as the PIP25 index, which are based on the relative concentrations of IP and sterols in Arctic marine sediment cores.