Long chain alkyl diols (LCDs) have been detected in a range of marine and lacustrine environments, as well as in several algal cultures. However, the identity of the producers, their preferred ecological niche and seasonality are uncertain. We applied a gene-based approach to determine the identity and abundance of Eustigmatophyceae 18S rRNA genes and compared the data with the distribution of LCDs in the water column of Lake Challa (East Africa). Gene-based analysis revealed three known and two novel Eustigmatophyceae groups. Maxima in the number of gene copies and LCD concentration coincided at 9 m water depth, signifying Eustigmatophyceae as important producers of LCDs. In addition, seasonal changes in LCD abundance in sedimenting particles revealed several blooms of LCD producers over the annual cycle.