Genomes are replicated in a reproducible temporal pattern. Current methods for assaying allele replication timing are time consuming and/or expensive. These include high-throughput sequencing which can be used to measure DNA copy number as a proxy for allele replication timing. Here, we use droplet digital PCR to study DNA replication timing at multiple loci in budding yeast and human cells. We establish that the method has temporal and spatial resolutions comparable to the high-throughput

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... ncing approaches, while being faster than alternative locus-specific methods. Furthermore, the approach is capable of allele discrimination. We apply this method to determine relative replication timing across timing transition zones in cultured human cells. Finally, multiple samples can be analysed in parallel, allowing us to rapidly screen kinetochore mutants for perturbation to centromere replication timing. Therefore, this approach is well suited to the study of locus-specific replication and the screening of cis- and trans-acting mutants to identify mechanisms that regulate local genome replication timing.