Ribonucleotide triphosphate (rNTP) incorporation into DNA in the form of ribonucleotide monophosphate (rNMP) is a common phenomenon in nature resulting in DNA structural change, genome instability, and alteration of protein-DNA interaction. Previous studies using the ribose-seq technique revealed abundant rNTP incorporation in mitochondrial DNA (mtDNA) of budding yeast Saccharomyces cerevisiae and green algae Chlamydomonas reinhardtii. However, the study of rNTP incorporation in human mtDNA has been limited to basic count and composition study in HeLa and fibroblast cells. The characteristics, hotspots, and association with the cellular process of rNTP incorporation remain to be discovered. Here, we utilized the ribose-seq technique to capture the incorporated rNTPs in mtDNA of 32 samples within 10 different human cell types. We identified a consistent rNTP-incorporation preference on the light strand in most cell types except the liver tissue cells. The rNMP hotspots and rNMP-enriched regions are also revealed in human mtDNA, including three conservative rNMP-enriched regions before the replication origin of the heavy strand, which may be related to human mtDNA replication. The compositions of incorporated rNTPs vary among the different cell types and are not necessarily related to the rNTP concentration in the cells, indicating that the rNTPs are not randomly incorporated into DNA. Moreover, by studying the genomic context of rNTP incorporation sites, we found that the upstream dNTPs have a strong impact on rNTP incorporation. Incorporation of rATP after dTMP, and rCTP after dCMP are preferred in all human 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 rNTP incorporation rate, which suggests a potential relationship between rNTP incorporation and gene expression in human mitochondria.