Abundant ribonucleotide triphosphates (rNTP) are incorporated into DNA by DNA polymerases in the form of ribonucleoside monophosphates (rNMPs). The incorporated rNMPs in DNA lead to DNA structural change, genome instability, and are associated with human diseases including Aicardi Goutieres syndrome and different types of cancer. In human mitochondrial DNA (hmtDNA), rNMPs are abundant due to the lack of the ribonucleotide excision repair pathway. The rNMP distribution, characteristics, hotspots, and preferred patterns in mtDNA of various human cell types remain to be discovered. In this study, we utilized the ribose-seq technique to capture rNMPs incorporated in the mtDNA of six different human cell types. We aligned the rNMPs to single-nucleotide coordinates in the hmtDNA reference genome using the Ribose-Map bioinformatics toolkit and performed analysis on the rNMP distribution, characteristics, hotspots, preferred patterns, and association with hmtDNA replication sites and genes. We also compared the sequence characteristics of rNTPs incorporated in hmtDNA with those found in yeast mtDNA. We found marked rNTP-incorporation preference on the light strand of hmtDNA in most cell types, but not in the liver tissue cells, which have the opposite strand preference on the heavy strand. We uncovered rNTP-enriched zones (REZs) in hmtDNA, and identified several conserved REZs across the different cell types, despite the ribonuclease-H genotype and the DNA fragmentation methods. Interestingly, we found three REZs in the hmtDNA replication control region. These REZs may affect the replication of hmtDNA by impairing the binding activity of hmtDNA polymerase Pol γ and helicase TWINKLE. Conserved REZs were also present in yeast mtDNA, suggesting that rNTP incorporation is not only far from being a random process but may also relate to structural and/or functional features of the mtDNA genomes. Furthermore, we located rNMP hotspots in hmtDNA of the different cell types and studied their features. We unveiled the association between rNTP-incorporation frequency and gene size. The longer coding sequences have a significantly higher rNTP-incorporation frequency at each of their nucleotides. While the composition of the incorporated rNMPs varied among the different cell types, by examining the genomic context of rNTP-incorporation sites, we detected common rNMP-incorporation patterns in hmtDNA. The genomic contexts of rNMPs found in hmtDNA were distinct from those found in yeast mtDNA, highlighting a unique signature of rNTP incorporation by hmtDNA Pol γ.