The sex pheromone component muscalure [(Z)-9-tricosene] on the cuticles of female house flies (Musca domestica L.) was quantified using gas chromatography. Laboratory and wild-strain house fly colonies were derived from poultry and dairy farms and a horse-riding stable. Muscalure levels from strain MB females were highest after 13 and 23 generations in culture, but 30–70% of females from early generations (F8–F12) had no detectable levels of muscalure. Flies <2 d old from the MB laboratory colony (F23) had less muscalure than did older flies, and levels tended to increase as flies aged from 3 to 8 d. Of 10 wild house fly populations sampled at 3 d of age (72–96 h old) between F1 and F7 in culture, 1 had no detectable muscalure, and 8 of 10 averaged ≤218 ng of muscalure per female, comprising ≤1.6% of total cuticular hydrocarbons. In 6 of 10 field populations, ≥67% of females had no detectable muscalure. In contrast, two field strains had average levels of 559 and 1,113 ng muscalure/female (comprising 3.2 and 5.0% of total hydrocarbons), which was comparable to long-established laboratory strains such as the UCR strain (751 ng muscalure/female, comprising 3.0% of total hydrocarbons). More than 70% of females from those two field strains had muscalure. Flies from the Cooper laboratory strain also lacked detectable muscalure. Muscalure amounts were highly variable; it was rare or lacking in many field populations and thus does not seem to be essential for house fly reproduction.