Several reports from mammals indicate that an increase in the mutation rate in 
late-replicating regions may, in part, be responsible for the observed genomic 
heterogeneity in neutral substitution rates and levels of diversity, although 
the mechanisms for this remain poorly understood. Recent evidence also suggests 
that late replication is associated with high mutability in yeast. This then 
raises the question as to whether a similar effect is operating across all 
eukaryotes. Limited evidence from one chromosome arm in Drosophila melanogaster 
suggests the opposite pattern, with regions overlapping early-firing origins 
showing increased levels of diversity and divergence. Given the availability of 
genome-wide replication timing profiles for D. melanogaster, we now return to 
this issue. Consistent with what is seen in other taxa, we find that divergence 
at synonymous sites in exon cores, as well as divergence at putatively 
unconstrained intronic sites, is elevated in late-replicating regions. Analysis 
of genes with low codon usage bias suggests a ∼30% difference in mutation rate 
between the earliest and the latest replicating sequence. Intronic sequence 
suggests a more modest difference. We additionally show that an increase in 
diversity in late-replicating sequences is not owing to replication timing 
covarying with the local recombination rate. If anything, the effects of 
recombination mask the impact of replication timing. We conclude that, contrary 
to prior reports and consistent with what is seen in mammals and yeast, there is 
indeed a relationship between rates of nucleotide divergence and diversity and 
replication timing that is consistent with an increase in the mutation rate 
during late S-phase in D. melanogaster. It is therefore plausible that such an 
effect might be common among eukaryotes. The result may have implications for 
the inference of positive selection.