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Attached growth bioprocesses that use biofilms to remove organic matter or nutrients from wastewater are known to harbor antibiotic resistance genes (ARGs). Biofilms in these processes are spatially heterogeneous, but little is known about depth stratification of ARGs in complex, mixed culture biofilms. To address this knowledge gap, we used an experimental approach combining cryosectioning and quantitative polymerase chain reaction to quantify the spatial distribution of three ARGs (sul1, ermB, and qnrS) and the class 1 integron‐integrase gene intI1 in biofilms from a lab‐scale rotating annular reactor fed with synthetic wastewater. We also used high throughput 16S ribosomal RNA (rRNA) gene sequencing to characterize community structure with depth in biofilms. The ARG sul1 and the integron‐integrase gene intI1 were found in higher abundances in upper layers of biofilm near the fluid‐biofilm interface …