Ribonucleotide (rNMP) incorporation in DNA is a common phenomenon in nature resulting in DNA structural change, genome instability, and alteration of protein-DNA interaction. Previous studies revealing abundant rNMP incorporation in human mitochondrial DNA (mtDNA) have been limited to HeLa and fibroblast cells. However, characteristics and hotspots of rNMP incorporation remain to be discovered. Here, we utilized the ribose-seq technique to capture the incorporated rNMPs in mtDNA of 32 samples within 10 different human cell types. We identified a consistent rNMP-incorporation preference on the light strand in most cell types except the liver tissue cells. The compositions of incorporated rNMPs vary among the different cell types and is not necessarly related to the rNMP concentration, indicating that the rNMPs are not randomly incorporated into DNA. Moreover, by studying the genomic context of rNMP incorporation sites, we found that the upstream dNMPs have a strong impact on rNMP incorporation. Incorporation of rAMP after dTMP, and rCMP after dCMP are preferred in all cell types. We identified other preferred patterns in one or more cell types as well, which may be related to the cell functions. We unveiled that the non-template strand of coding sequences has a significantly higher rNMP incorporation rate, which suggests a potential relationship between rNMP incorporation and gene expression in human mitochondria. We also located hotspots sites and hotspot regions common to all the cell types, including two consistent peaks in the mtDNA replication control region, suggesting the potential relation to the mtDNA replication process.