Simple SummaryCircadian clocks are molecular pacemakers that drive daily rhythms in physiology, metabolism, and behavior. They are present in a diverse range of organisms, from cyanobacteria to humans. In the last few decades, enormous progress has been made in understanding the molecular details of the circadian clock. The fruit-flyDrosophilahas been instrumental in identifying ‘clock’ genes, which are well conserved in mammals, both in sequence and function. Many ‘clock’ genes have been identified using laboratory mutagenesis screens, which look for mutants that show aberrant circadian behavior. The recent accumulation of genomic sequences from wild populations ofDrosophilaprovides an opportunity to identify natural genetic variations in ‘clock’ genes, which serve as molecular adaptations to local habitats. Here, we focused on nine polymorphic sites in genes associated with the clock and tested their impact on diurnal behavior. We found seven sites whose alleles confer different circadian behavior or seasonal response. This analysis of natural variation can provide information about fine-tuning of the clock, and it is essential for understanding the evolution of the circadian system.AbstractOur understanding of the gene regulatory network that constitutes the circadian clock has greatly increased in recent decades, notably due to the use ofDrosophilaas a model system. In contrast, the analysis of natural genetic variation that enables the robust function of the clock under a broad range of environments has developed more slowly. In the current study, we analyzed comprehensive genome sequencing data from wild European populations ofDrosophila, which were densely sampled through time and space. We identified hundreds of single nucleotide polymorphisms (SNPs) in nine genes associated with the clock, 276 of which exhibited a latitudinal cline in their allele frequencies. While the effect sizes of these clinal patterns were small, indicating subtle adaptations driven by natural selection, they provided important insights into the genetic dynamics of circadian rhythms in natural populations. We selected nine SNPs in different genes and assessed their impact on circadian and seasonal phenotypes by reconstructing outbred populations fixed for either of the SNP alleles, from inbred DGRP strains. The circadian free-running period of the locomotor activity rhythm was affected by an SNP indoubletime(dbt) andeyes absent(Eya). The SNPs inClock(Clk),Shaggy(Sgg),period(per), andtimeless(tim) affected the acrophase. The alleles of the SNP inEyaconferred different levels of diapause and the chill coma recovery response.