Activated sludge has been identified as a potential significant source of antibiotic resistance genes (ARGs) to the environment. Anaerobic digestion is extensively used for sludge stabilization and resource recovery, and represents a crucial process for controlling the dissemination of ARGs prior to land application of digested sludge. The objective of this study is to investigate the effect of zero valent iron (Fe0) on the attenuation of seven representative tetracycline resistance genes (tet, tet(A), tet(C), tet(G), tet(M), tet(O), tet(W), and tet(X)), and the integrase gene intI1 during thermophilic anaerobic co-digestion of waste sludge and kitchen waste. Significant decrease (P < 0.05) in the quantities of tet (except tet(W)) and intI1 genes was observed at Fe0 dosage of 5 g/L, whereas no significant differences (P > 0.05) were found for all gene targets between digesters with Fe0 dosages of 5 and 60 g/L. A first-order kinetic model favorably described the trends in concentrations of tet and intI1 gene targets during thermophilic anaerobic digestion with or without Fe0. Notably, tet genes encoding different resistance mechanisms behaved distinctly in anaerobic digesters, although addition of Fe0 could enhance their reduction. The overall results of this research suggest that thermophilic anaerobic digestion with Fe0 can be a potential alternative technology for the attenuation of tet and intI1 genes in waste sludge.