Lipids are an important class of nutrients for insects, as they are the main energy source during metamorphosis and crucial for survival and reproduction. Lipid reserves in insects are mostly accumulated during larval development and can be influenced by environmental factors such as diet and temperature. Genetic factors can also affect larval lipid content and composition. Larvae of the house fly, Musca domestica L. (Diptera: Muscidae), are a valuable emerging resource for the insect feed sector, fueling a growing interest in the factors regulating larval lipid content. We investigated whether strains of three geographical origins vary in ontogeny, quantity, and composition of stored lipids in response to temperature. Italian, Spanish, and Dutch house fly larvae were reared on a similar diet at 25 and 35 °C. Larval dry weight and total lipid content were determined in early‐, mid‐, and late‐third instars by extraction of soluble lipids. The fatty acid composition was analyzed in late‐third instars using an improved extraction and derivatization protocol. We found that strain, larval stage, and temperature affected larval dry weight and absolute and relative lipid content, with significant interaction effects between these three factors. All flies were reared in a common environment, indicating a genetic component to lipid storage. Analysis of lipid composition identified 11 fatty acids, including some rarely reported in the literature. Five fatty acids accounted for 80 and 81% of the total fatty acid methyl esters detected at 25 and 35 °C, respectively. An effect of temperature but not strain was evident on the composition of the fatty acids. The observed differences in lipid content among strains and temperature conditions could be of interest for commercial rearing of the house fly.