Amphibians generally have genetic sex determination which should lead to equal sex ratios. However, populations of the alpine newt (Mesotriton alpestris) from cold, high-altitude breeding sites in the Austrian Alps showed strongly female-biased sex ratios of up to 3.7 females to 1 male over 6 years. Temperature dependent sex determination has therefore been considered as possible reason to explain the observed skews. To investigate if a female excess already exists during gonadal differentiation of M. alpestris larvae before metamorphosis, we assessed larval sex ratios in high-altitude spawning sites with different temperature regimes. The sex was determined by histological analyses of cross sections of the gonads. In no case a significant divergence from a 1:1 sex ratio was found. Based on these results and previous karyological, skeletochronological, and ecological work we conclude that higher mortality rates of adult males are the most plausible explanation for the female excess in high-altitude populations of alpine newts. However, future studies should focus on the development of sex-specific genetic markers to allow early determination of gender in embryos and larvae.