Genomic DNA replicates in a choreographed temporal order that impacts the distribution of mutations along the genome. We show here that DNA replication timing is shaped by genetic polymorphisms that act in cis upon megabase-scale DNA segments. In genome sequences from proliferating cells, read depth along chromosomes reflected DNA replication activity in those cells. We used this relationship to analyze variation in replication timing among 161 individuals sequenced by the 1000 Genomes Project. Genome-wide association of replication timing with genetic variation identified 16 loci at which inherited alleles associate with replication timing. We call these “replication timing quantitative trait loci” (rtQTLs). rtQTLs involved the differential use of replication origins, exhibited allele-specific effects on replication timing, and associated with gene expression variation at megabase scales. Our results show replication timing to be shaped by genetic polymorphism and identify a means by which inherited polymorphism regulates the mutability of nearby sequences. Display omitted •Replication timing, a driver of locus-specific mutation rates, varies among humans•Whole genome sequence data can be used to study DNA replication activity•Replication timing associates with common polymorphisms near replication origins•Replication timing QTLs have megabase-scale effects on replication and transcription Replication timing varies among humans, and genetic variants associate with replication timing. These replication timing quantitative trait loci are cis-acting modifiers of replication timing, gene expression levels, and local mutation rates and are an unexpected source of potential wide-reaching variation between individuals.