Long-chain alkenones, unique class of lipid biomarker produced by members of the Isochrysidales haptophytes, have been widely used for reconstructing past sea surface temperature (SST) from marine sediment cores. In recent decades, alkenones have also been discovered from some freshwater and saline lakes. The application of alkenone paleothermometer in lakes and some marginal seas are often hindered by the potential occurrence of species different from the typical marine alkenone producers: different species often exhibit significantly different temperature calibrations. Recent molecular biological studies updated the overall view of diversity in the alkenone producers, dividing Isochrysidales into three major groups: Group I consists of uncultured sequences occur in freshwater lakes, Group II includes Isochrysidaceae haptophytes often found in saline lakes and marginal sea, and Group III consists of Noëlaerhabdaceae haptophytes (ocean species).Laboratory culture experiments of Group II species highlight significant chemotaxonomic variations in both biomarker compositions and temperature calibrations, reflecting a wider phylogenetic diversity compared to Group III. Recent field studies also suggest the conserved characteristics in alkenone and temperature responses in Group I haptophytes. A combination of environmental DNA analysis and chemotaxonomic profiling is essential for the identification of alkenone producers and improved applications of alkenone-based indices for paleoclimate and paleoenvironmental research.