Reshuffling of genetic variation occurs both by independent assortment of chromosomes and by homologous recombination. Such reshuffling can generate novel allele combinations and break linkage between advantageous and deleterious variants which increases both the potential and the efficacy of natural selection. Here we used high-density linkage maps to characterize global and regional recombination rate variation in two populations of the wood white butterfly (Leptidea sinapis) that differ considerably in their karyotype as a consequence of at least 27 chromosome fissions and fusions. The recombination data were compared to estimates of genetic diversity and measures of selection to assess the relationship between chromosomal rearrangements, crossing over, maintenance of genetic diversity and adaptation. Our data show that the recombination rate is influenced by both chromosome size and number, but that the difference in the number of crossovers between karyotypes is reduced as a consequence of a higher frequency of double crossovers in larger chromosomes. As expected from effects of selection on linked sites, we observed an overall positive association between recombination rate and genetic diversity in both populations. Our results also revealed a significant effect of chromosomal rearrangements on the rate of intergenic diversity change between populations, but limited effects on polymorphisms in coding sequence. We conclude that chromosomal rearrangements can have considerable effects on the recombination landscape and consequently influence both maintenance of genetic diversity and efficiency of selection in natural populations. Author summaryReshuffling genetic variation is fundamental for maintaining genetic diversity and creating novel allelic combinations. The two main processes involved are the independent assortment of chromosomes and homologous recombination. The number and size of chromosomes can influence the amount of pairwise reshuffling and local recombination patterns. However, studying this in natural populations is challenging. In this study, we used the wood white butterfly, which exhibits an extreme within-species karyotype difference. Extensive fusions and fissions have resulted in almost twice as many chromosomes in the southern populations compared to the northeast populations. This unique system allowed us to assess the relationship between karyotype differences, pairwise reshuffling, recombination rate variation and subsequent effects on diversity and linked selection. We found that a higher number of chromosomes results in higher number of crossovers, although the difference was less than expected due to multiple recombination events occurring on longer chromosomes. Both populations showed an association between recombination rate and genome-wide patterns of genetic diversity and efficacy of selection. We provide evidence that chromosomal rearrangements have considerable effects on the recombination landscape and thereby influence the maintenance of genetic diversity in populations.