Adeno-associated virus (AAV) gene therapy for neurological diseases was revolutionized by the discovery that AAV9 crosses the blood-brain barrier (BBB) after systemic administration. Transformative results have been documented in various inherited diseases, but overall neuronal transduction efficiency is relatively low. The recent development of AAV-PHP.B with ∼60-fold higher efficiency than AAV9 in transducing the adult mouse brain was the major first step toward acquiring the ability to deliver genes to the majority of cells in the central nervous system (CNS). However, little is known about the mechanism utilized by AAV to cross the BBB, and how it may diverge across species. In this study, we show that AAV-PHP.B is ineffective for systemic CNS gene transfer in the inbred strains BALB/cJ, BALB/cByJ, A/J, NOD/ShiLtJ, NZO/HILtJ, C3H/HeJ, and CBA/J mice, but it is highly potent in C57BL/6J, FVB/NJ, DBA/2J, 129S1/SvImJ, and AKR/J mice and also the outbred strain CD-1. We used the power of classical genetics to uncover the molecular mechanisms AAV-PHP.B engages to transduce CNS at high efficiency, and by quantitative trait locus mapping we identify a 6 Mb region in chromosome 15 with an logarithm of the odds (LOD) score ∼20, including single nucleotide polymorphisms in the coding region of 9 different genes. Comparison of the publicly available data on the genome sequence of 16 different mouse strains, combined with RNA-seq data analysis of brain microcapillary endothelia, led us to conclude that the expression level of