The characterization of native polycyclic aromatic hydrocarbon (PAH)-degrading bacteria is significant for understanding the PAH degradation process in the natural environment and developing effective remediation technologies. Most previous investigations of PAH-degrading bacteria in environmental samples employ pahAc, which encodes the α-subunit of PAH ring-hydroxylating dioxygenase, as a functional marker gene. However, the poor phylogenetic resolution and nonspecificity of pahAc result in a misestimation of PAH-degrading bacteria. Here, we propose a PAH hydratase-aldolase-encoding gene, pahE, as a superior biomarker for PAH-degrading bacteria. Comparative phylogenetic analysis of the key enzymes involved in the upper pathway of PAH degradation indicated that pahE evolved dependently from a common ancestor. A phylogenetic tree constructed based on PahE is largely congruent with PahAc-based phylogenies, except for the dispersion of several clades of other non-PAH-degrading aromatic hydrocarbon dioxygenases present in the PahAc tree. Analysis of pure strains by PCR confirmed that pahE can specifically distinguish PAH-degrading bacteria, while pahAc cannot. Illumina sequencing of pahE and pahAc amplicons showed more genotypes and higher specificity and resolution for pahE Novel reads were also discovered among the pahE amplicons, suggesting the presence of novel PAH-degrading populations. These results suggest that pahE is a more powerful biomarker for exploring the ecological role and degradation potential of PAH-degrading bacteria in ecosystems, which is significant to the bioremediation of PAH pollution and environmental microbial ecology.IMPORTANCE PAH contamination has become a worldwide environmental issue because of the potential toxic effects on natural ecosystems and human health. Biotransformation and biodegradation are considered the main natural elimination forms of PAHs from contaminated sites. Therefore, the knowledge of the degradation potential of the microbial community in contaminated sites is crucial for PAH pollution bioremediation. However, the nonspecificity of pahAc as a functional marker of PAH-degrading bacteria has resulted neither in a reliable prediction of PAH degradation potential nor an accurate assessment of degradation. Here, we introduced pahE encoding the PAH hydratase-aldolase as a new and better functional marker gene of PAH-degrading bacteria. This study provides a powerful molecular tool to more effectively explore the ecological role and degradation potential of PAH-degrading bacteria in ecosystems, which is significant to the bioremediation of PAH pollution.