The evolutionary response to regional and global climate change may vary in widespread polymorphic species, so predicting future genetic responses will require careful tracking of genetic variability in local populations. We surveyed chromosomal inversion polymorphisms in 25 populations of Drosophila robusta, many of which have been sampled repeatedly starting in the 1940s, 50s, and 60s up until 2007, across its range in the USA. Frequencies of some northerly, or cold-adapted, gene arrangements have declined in the face of increasing temperatures, whereas frequencies of several southern, or warm-adapted, gene arrangements were positively correlated with increasing temperature changes. Over a finer geographic scale, populations from the west-central part of the species range from the Ozark Plateau, Ouachita mountains, and eastern Oklahoma showed genetic differentiation between south-central Ozark and western Ozark/Ouachita regions that has persisted in the face of recent shifts in gene arrangement frequencies. Overall, populations of D. robusta exhibited dynamic genetic changes over time, with some populations shifting chromosome frequencies in just 10–15 years. Some temporal genetic shifts were widespread and significantly correlated with temperature increases, but regions of the genome marked by different gene arrangements have responded in different sections of the species range. In some parts of the species range, chromosome frequencies shifted but were not associated with changing temperatures, showed little or no temporal change, or temporal shifts stopped for temperature sensitive gene arrangements near fixation. © 2008 The Linnean Society of London, Biological Journal of the Linnean Society, 2008, 95, 702–718.