Whole exome sequencing (WES) has been used to detect rare causative variants in neurological diseases. However, the efficacy of WES in genetic diagnosis of clinically heterogeneous familial stroke remains inconclusive. We prospectively searched for disease-causing variants in unrelated probands with defined familial stroke by candidate gene/hotspot screening and/or WES, depending on stroke subtypes and neuroimaging features at a referral center. The clinical significance of each variant was determined according to the American College of Medical Genetics guidelines. Among 161 probands (mean age at onset 53.2 ± 13.7 years; male 63.4%), 33 participants (20.5%) had been identified with 19 pathogenic/likely pathogenic variants (PVs; WES applied 152/161 = 94.4%). Across subtypes, the highest hit rate (HR) was intracerebral hemorrhage (ICH, 7/18 = 38.9%), particularly with the etiological subtype of structural vasculopathy (4/4 = 100%, PVs inENG, KRIT1, PKD1, RNF213); followed by ischemic small vessel disease (SVD, 15/48 = 31.3%; PVs inNOTCH3, HTRA1, HBB). In contrast, large artery atherosclerosis (LAA, 4/44 = 9.1%) and cardioembolism (0/11 = 0%) had the lowest HR.NOTCH3was the most common causative gene (16/161 = 9.9%), presenting with multiple subtypes of SVD (n= 13), ICH (n= 2), or LAA (n= 1). Importantly, we disclosed two previously unreported PVs,KRIT1p.E379* in a familial cerebral cavernous malformation, andF2p.F382L in a familial cerebral venous sinus thrombosis. The contribution of monogenic etiologies was particularly high in familial ICH and SVD subtypes in our Taiwanese cohort. Utilizing subtype-guided hotspot screening and/or subsequent WES, we unraveled monogenic causes in 20.5% familial stroke probands, including 1.2% novel PVs. Genetic diagnosis may enable early diagnosis, management and lifestyle modification.Graphical AbstractAmong 161 familial stroke probands, 33 (20.5%) had been identified pathogenic or likely pathogenic monogenic variants related to stroke. The positive hit rate among all subtypes was high in intracerebral hemorrhage (ICH) and ischemic small vessel disease (SVD). Notably, two previously unreported variants,KRIT1p.E379* in a familial cerebral cavernous malformation andF2p.F382L in familial cerebral venous sinus thrombosis, were disclosed.CVTcerebral venous thrombosis;HTNHypertensive subtype;LAAlarge artery atherosclerosis;SVstructural vasculopathy;UUndetermined.Supplementary InformationThe online version contains supplementary material available at 10.1007/s10571-022-01315-3.