Male but not female mice carrying a single R403Q missense allele for cardiac α-myosin heavy chain (M-αMHC^R403Q/+ and F-αMHC^R403Q/+, respectively) develop significant hypertrophic cardiomyopathy (HCM) compared with male and female wild-type mice (M-αMHC^+/+ and F-αMHC^+/+, respectively) after ∼30 wk of age. We tested the hypothesis that myofilament mechanical performance differs between M-αMHC^R403Q/+ and F-αMHC^R403Q/+ at younger ages (10–20 wk) and could account for sex differences in HCM development. The sensitivity of chemically skinned myocardial strips to Ca²⁺ activation (pCa₅₀) was significantly (P < 0.05) enhanced in male mice independent of genotype (M-αMHC^R403Q/+: 5.70 ± 0.06, M-αMHC^+/+: 5.63 ± 0.05, F-αMHC^R403Q/+: 5.57 ± 0.03, F-αMHC^+/+: 5.54 ± 0.04) by two-way ANOVA, whereas maximum developed tension was significantly enhanced in α-MHC^R403Q/+ independent of sex (M-αMHC^R403Q/+: 29.3 ± 2.3, M-αMHC^+/+: 26.0 ± 1.4, F-αMHC^R403Q/+: 30.2 ± 2.1, F-αMHC^+/+: 26.2 ± 1.2 mN/mm²). The frequency of maximum work generated by sinusoidal length perturbation was significantly higher in αMHC^R403Q/+ mice than in sex-matched controls (M-αMHC^R403Q/+: 2.26 ± 0.47, M-αMHC^+/+: 1.29 ± 0.18, F-αMHC^R403Q/+: 3.21 ± 0.33, F-αMHC^+/+: 2.52 ± 0.36 Hz). Unloaded shortening velocity was significantly enhanced in αMHC^R403Q/+ and in female mice (M-αMHC^R403Q/+: 2.26 ± 0.47, M-αMHC^+/+: 1.29 ± 0.18, F-αMHC^R403Q/+: 3.21 ± 0.33, F-αMHC^+/+: 2.52 ± 0.36 muscle lengths/s), and normalized mechanical power, calculated from the tension-velocity relationship, was significantly enhanced in αMHC^R403Q/+ independent of sex (M-αMHC^R403Q/+: 60 ± 2 10⁻³, M-αMHC^+/+: 37 ± 3 10⁻³, F-αMHC^R403Q/+: 57 ± 3 10⁻³, F-αMHC^+/+ 25 ± 3 10⁻³ muscle lengths/s × normalized tension). We did not find a statistically significant sex × mutation interaction for any measure of myofilament performance. Therefore, sarcomeric incorporation of the R403Q myosin similarly enhanced left ventricular myofilament mechanical performance in both male and female mice. The sex-dependent development of HCM due to the R403Q myosin may then be inhibited by female sex hormones, which may additionally underlie the observed sex differences for pCa₅₀ and unloaded shortening velocity.