AbstractA barrier to advancing engineered adeno-associated viral vectors (AAVs) for precision access to cell subtypes is a lack of high-throughput, high-resolution assays to characterize in vivo transduction profiles. In this study, we developed an ultrasensitive, sequential fluorescence in situ hybridization (USeqFISH) method for spatial transcriptomic profiling of endogenous and viral RNA with a short barcode in intact tissue volumes by integrating hydrogel-based tissue clearing, enhanced signal amplification and multiplexing using sequential labeling. Using USeqFISH, we investigated the transduction and cell subtype tropisms across mouse brain regions of six systemic AAVs, including AAV-PHP.AX, a new variant that transduces robustly and efficiently across neurons and astrocytes. Here we reveal distinct cell subtype biases of each AAV variant, including a bias of AAV-PHP.N toward excitatory neurons. USeqFISH also enables profiling of pooled regulatory cargos, as we show for a 13-variant pool of microRNA target sites in AAV genomes. Lastly, we demonstrate potential applications of USeqFISH for in situ AAV profiling and multimodal single-cell analysis in non-human primates.