Identifying functional microorganisms involved in the degradation of high-molecular-mass polycyclic aromatic hydrocarbons (HMM-PAHs) in agricultural soil environments could assist in developing bioremediation strategies for soil PAH contamination. Active populations of HMM-PAH degraders in agricultural soils are currently poorly understood. In this study, we identified aerobic pyrene-degrading bacteria in agricultural and industrial soils by [13C]pyrene incubations followed by DNA stable-isotope probing and high-throughput sequencing. More than 80% of pyrene was degraded during an incubation time of 35 days in both soils, with slower mineralization rates observed in agricultural soil compared with industrial soil. Members of the Pseudonocardia genus, not previously implicated in pyrene degradation, were the dominant pyrene-degrading population in agricultural soil; their relative abundance increased by three orders of magnitude. In industrial soil, Arthrobacter sp. appeared as the major pyrene degraders, while Pseudonocardia was not detectable. Mycobacterium, a group of well-known pyrene degraders, was found to be active in pyrene degradation in both soils. These results highlight the role of uncultivated members of Pseudonocardia in natural PAH biodegradation processes and expand our understanding of the metabolic potential of uncultivated microorganisms for bioremediation applications in agricultural soils.