Murine gammaherpesvirus 68 (MHV-68) replicates robustly in cell culture, providing a model for studying viral genome replication during de novo infection of tumor-associated herpesviruses. We have previously identified a 1.25-kb origin of lytic replication (oriLyt) for MHV-68. To further investigate the molecular mechanism of viral genome replication, we first fine-mapped essential cis-elements from this oriLyt fragment using a transposon-mediated high-density mutagenesis method. The result provided information for us to identify a second oriLyt located towards the left end of MHV-68 genome using a de novo infection-replication assay. We further characterized this left oriLyt by scanning deletion analysis and site-directed mutations, and showed that several CCAAT motifs are essential for oriLyt function, whereas an AT-rich region enhances replication. However, GC-rich repeats are not important cis-element. Moreover, we identified a cellular transcription factor, NF-Y, which binds to CCAAT boxes in EMSA and associates with oriLyt in ChIP assay. Using a dominant negative expression plasmid, we demonstrated that NF-Y plays an important role in mediating MHV-68 genome replication during de novo infection.