Three eukaryotic DNA polymerases are essential for genome replication. Polymerase (Pol) -primase initiates each synthesis event and is rapidly replaced by processive DNA polymerases: Polε replicates the leading strand, whereas Polδperforms lagging-strand synthesis. However, it is not known whether this division of labor is maintained across the whole genome or how uniform it is within single replicons. Using Schizosaccharomyces pombe, we have developed a polymerase usage sequencing (Pu-seq) strategy to map polymerase usage genome wide. Pu-seq provides direct replication-origin location and efficiency data and indirect estimates of replication timing. We confirm that the division of labor is broadly maintained across an entire genome. However, our data suggest a subtle variability in the usage of the two polymerases within individual replicons. We propose that this results from occasional leading-strand initiation by Polδfollowed by exchange for Polε.