Alcohol septal ablation (ASA) is performed for symptomatic drug-refractory hypertrophic obstructive cardiomyopathy to reduce the left ventricular outflow tract pressure gradient (LVOTPG) by injecting ethanol into a septal branch that perforates the septal bulge. The target septal branches usually arise directly from the left anterior descending (LAD) artery; however, vessels from a non-LAD artery can be selected in some cases. This study aimed to compare effectiveness and safety between ASA performed using a septal branch arising from a non-LAD artery and a branch arising from the LAD artery. This single-center, retrospective, observational cohort study comprised patients with hypertrophic obstructive cardiomyopathy who underwent ASA at the Gifu Heart Centre between 2011 and 2022. The effectiveness and safety of ASA using the 2 artery types were compared. The primary endpoints were LVOTPG and procedure success, determined as LVOTPG <30 mmHg after 1 year. Of 33 patients (mean age: 66.4 ± 13.0 years; 13 males), 18 patients who underwent ASA using only LAD branches and 15 patients who underwent ASA using only non-LAD branches demonstrated no significant difference in the decrease in LVOTPG during the follow-up period (−99.1 ± 47.4 mmHg/year vs. −75.7 ± 39.2 mmHg/year, respectively, P = 0.19). The procedure success at 1 year was not significantly different between the 2 groups (93.3% and 84.6%, respectively, P = 0.58). ASA performed using septal branches from non-LAD arteries could be an alternative treatment approach when appropriate septal branches are missing or desirable effects cannot be obtained from ASA using LAD branches.