Handedness in humans – better performance using either the left or right hand – is personally familiar, moderately heritable1, and regulated by many genes2, including those involved in general body symmetry3. But behavioral handedness, i.e. lateralization, is a multifaceted phenomenon. For example, people display clockwise or counter-clockwise biases in their walking behavior that is uncorrelated to their hand dominance4,5, and lateralized behavioral biases have been shown in species as disparate as mice (paw usage6), octopi (eye usage7), and tortoises (side rolled on during righting8). However, the mechanisms by which asymmetries are instilled in behavior are unknown, and a system for studying behavioral handedness in a genetically tractable model system is needed. Here we show that Drosophila melanogaster flies exhibit striking variability in their left-right choice behavior during locomotion. Very strongly biased "left-handed" and "right-handed" individuals are common in every line assayed. The handedness of an individual persists for its lifetime, but is not passed on to progeny, suggesting that mechanisms other than genetics determine individual handedness. We use the Drosophila transgenic toolkit to map a specific set of neurons within the central complex that regulates the strength of behavioral handedness within a line. These findings give insights into choice behaviors and laterality in a simple model organism, and demonstrate that individuals from isogenic populations reared under experimentally identical conditions nevertheless display idiosyncratic behaviors.