This study systematically evaluated five microbial and four mitochondrial DNA (mtDNA) markers, including sensitivities and specificities under PCR method, and fecal concentrations and decay rates in water under qPCR method. The microbial DNA markers were the three human-associated (BacH, HF183 and B.adolescentis) and two pig-associated (Pig-2-Bac and L.amylovorus), while the mtDNA ones were two human- (H-ND6 and H-ND5) and two pig-associated (P-CytB and P-ND5). All the mtDNA markers showed higher sensitivity (100%) than the microbial ones (84.0-88.8%) except Pig-2-Bac (100%). Specificities of the human mtDNA markers (99.1 and 98.1%) were higher than those of the human-associated microbial ones (57.0-88.8%). But this pattern was not observed in the pig-associated markers where Pig-2-Bac had 100% specificity. The reliability of H-ND6 and H-ND5 was further evidenced to identify locations of the most polluted within the Taihu Lake watershed of China. In general, the microbial DNA markers demonstrated a higher fecal concentration than the mtDNA ones; increasing temperature and sunlight exposure accelerated significantly the decay of all the DNA markers. Results of this study suggest that DNA markers H-ND6, H-ND5, and Pig-2-Bac may be among the best for fecal source tracking in water.