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Ectotherms decrease in size with increasing ambient temperature. Temperature–size relationships (TSR) have been observed experimentally in a wide range of animals, algae, protozoans and bacteria. However, it is still unclear whether temperature is an important factor controlling the size of organisms in natural populations. In this study, we used natural variability in water temperature in the nearshore areas of a single lake to test TSR in populations of benthic diatoms. We deployed standard tile substrates at 5 m depth (similar light availability) at cold and warm sites that were exposed to different hydrodynamic forces. We compared cell sizes of three species of diatoms (Achnanthidium minutissimum, Gomphonema acuminatum and Gyrosigma acuminatum) at these sites. Counter to the TSR, diatom cells at warm sites were either larger (Achnanthidium, Gomphonema) or similar in size (Gyrosigma) compared to those at colder sites. Diatom size was also related to site exposure (hydrodynamic forces), but differently for species with different architectures. TSR were not detectable in the field for these three species of benthic diatom, even when tested within a single ecosystem at a given time of the year. The size of benthic diatoms, however, varied in a predictable way between sites, and such differences could affect the functioning of these primary producers in different parts of the littoral zone.