Replication timing is an important determinant of germline mutation patterns, with a higher rate of point mutations in late replicating regions. Mechanisms underlying this association remain elusive. One of the suggested explanations is the activity of error-prone DNA polymerases in late-replicating regions. Polymerase zeta (pol Ξ), an essential error-prone polymerase biased toward transversions, also has a tendency to produce dinucleotide mutations (DNMs), complex mutational events that simultaneously affect two adjacent nucleotides. Experimental studies have shown that pol Ξ is strongly biased toward GC→AA/TT DNMs. Using primate divergence data, we show that the GC→AA/TT pol Ξ mutational signature is the most frequent among DNMs, and its rate exceeds the mean rate of other DNM types by a factor of approximately 10. Unlike the overall rate of DNMs, the pol Ξ signature drastically increases with the replication time in the human genome. Finally, the pol Ξ signature is enriched in transcribed regions, and there is a strong prevalence of GC→TT over GC→AA DNMs on the nontemplate strand, indicating association with transcription. A recurrently occurring GC→TT DNM inHRASandSOD1genes causes the Costello syndrome and amyotrophic lateral sclerosis correspondently; we observe an approximately 1 kb long mutation hotspot enriched by transversions near these DNMs in both cases, suggesting a link between these diseases and pol Ξ activity. This study uncovers the genomic preferences of pol Ξ, shedding light on a novel cause of mutational heterogeneity along the genome.