Sexual isolation is a key component of reproductive isolation, involving mate choice among mature adults. While there are various statistics for estimating sexual isolation from mating frequencies, their ability to produce unbiased estimates varies considerably, depending on the particular situation. We investigated, under different biological scenarios, the estimation properties (statistical bias, efficiency, root mean square, statistical test) of 12 statistics commonly used in the literature for measuring sexual isolation. Yule’s Q, V, YA (and related indices) and IPSI are revealed to be the most efficient, with the smallest biases and root mean square deviations. Yule’s Q, YA and IPSI show better estimation properties when using infinite sample sizes, while IPSI is preferable using smaller sample sizes. Other statistics investigated should be avoided, at least within the range of conditions considered. Regarding the parametric test of hypothesis, the best alternative is YA. We discuss the advantages and drawbacks of the various estimators, and propose IPSI as the safest for biological sample sizes.