Background Despite causing up to a quarter of all strokes, the genetic basis of lacunar stroke remains poorly understood, with a single locus on 16q24 identified to date. We performed a genome-wide association study (GWAS) of lacunar stroke, expanding the sample size by recruiting robustly phenotyped cases using MRI in diagnosis, to identify novel associations and provide mechanistic insights into the disease. Methods We performed a GWAS of 7,338 cases and 225,258 controls, including newly recruited patients and reanalysis of existing studies, of which 2,987 cases (matched with 29,540 controls) were confirmed using MRI. We used multi-trait analysis of GWAS (MTAG), performing a joint analysis with a study of cerebral white matter hyperintensities (N=42,310) - an etiologically related radiological trait, to uncover additional genetic associations. We performed a transcriptome-wide association study (TWAS), to determine genes for which expression is associated with lacunar stroke, identified significantly enriched pathways using MAGMA and determined cardiovascular risk factors causally associated with the disease using Mendelian Randomization. Findings 5 loci were associated with lacunar stroke in European or Transethnic meta-analysis. A further 7 loci were associated in multi-trait analysis. Two loci contain genes (COL4A2 and HTRA1) involved in monogenic lacunar stroke. Pathway analyses implicated disruption of the extracellular matrix, phosphatidylinositol 5 phosphate binding, and roundabout (ROBO) binding at FDR<0.05, while Mendelian randomization linked elevated blood pressure, history of smoking, and type 2 diabetes in the etiology of lacunar stroke. Interpretation Lacunar stroke has a substantial heritable component, with 12 loci now identified that may represent future treatment targets. These loci provide insights into lacunar stroke pathogenesis, highlighting disruption of the vascular ECM (COL4A2, LOX, SH3PXD2A, GPR126, HTRA1), pericyte differentiation (FOXF2, GPR126), TGF-beta signaling (HTRA1), and myelination (ULK4, GPR126) in disease risk.