There is an active debate concerning the association of handedness and spatial ability. Past studies used small sample sizes within a single country. Determining the effect of handedness on spatial ability requires a large, cross-cultural sample of participants, and a navigation task with real-world validity. Here, we overcome these challenges via the mobile app Sea Hero Quest. We analysed the navigation performance from 422,772 participants from 41 countries and found no reliable evidence for any difference in spatial ability between left- and right-handers across all countries. Using 749,037 participants from the larger sample, we replicate previous findings that age, gender, and country of residence have an impact on the prevalence of left-handedness, and found an effect of education on left-handedness prevalence in China, Indonesia, India, Taiwan, and Hong Kong. Our study clarifies the factors associated with spatial ability and outlines new ways in which cultural patterns influence handedness.