Aims: The objective of this study was to determine whether varying levels of urbanization influence the dominant bacterial species of mildly resistant (0.03 mmol l(-1) tetracycline) and highly resistant (0.06 mmol l(-1) tetracycline) bacteria in sediment and water. Also, the level of urbanization was further evaluated to determine whether the diversity of tetracycline resistance genes present in the isolates and the capability of transferring their resistance were influenced. Methods and Results: Sediment and water samples collected from five sampling sites were plated in triplicate on nutrient agar plates with a mild dose (0.03 mmol l(-1)) and a high dose (0.06 mmol l(-1)) of tetracycline. Five colonies from each plate plus an additional five from each triplicate group were randomly selected and isolated on nutrient agar containing 0.03 mmol l(-1) tetracycline (400 isolates). The isolates were identified by 16S rRNA gene sequencing and comparison to GenBank using blast. The isolates were also screened for 15 tetracycline resistance genes using a multiplex PCR assay and their ability to transfer resistance through conjugation experiments using a kanamycin-resistant Escherichia. coli K-12 strain labelled with a green fluorescent protein gene. Results from this study indicate that the dominant resistant organisms in this watershed are Acinetobacter spp., Chryseobacterium spp., Serratia spp., Pseudomonas spp., Aeromonas spp. and E.coli. All of these organisms are Gram negative and are closely related to pathogenic species. A majority of the isolates (66%) were capable of transferring their resistance, and there was a greater incidence of tet resistance transfer with increasing urbanization. Also, it was determined that the dominant resistance genes in the watershed are tet(W) and tet(A). Conclusion: Urbanization significantly affected dominant tetracycline-resistant bacteria species, but did not affect dominant resistance genes. There was correlation between increased urbanization with an increase in the ability to transfer tetracycline resistance. This indicates that urban areas may select for bacterial species that are capable of transferring resistance. Significance and Impact of Study: These results indicate that urbanization influences the occurrence of tetracycline-resistant bacteria and the potential for transfer of resistance genes.