Transcriptional events during S-phase are critical for cell cycle progression. Here, by using a nascent RNA capture assay coupled with high-throughput sequencing, we determined the temporal patterns of transcriptional events that occur during S-phase. We show that genes involved in critical S-phase-specific biological processes such as nucleosome assembly and DNA repair have temporal transcription patterns across S-phase that are not evident from total RNA levels. By comparing transcription timing with replication timing in S-phase, we show that early replicating genes show increased transcription late in S-phase whereas late replicating genes are predominantly transcribed early in S-phase. Global anti-correlation between replication and transcription timing was observed only based on nascent RNA but not total RNA. Our data provides a detailed view of ongoing transcriptional events during the S-phase of cell cycle, and supports that transcription and replication are temporally separated.