Purpose: Chronic anal fissures are associated with significant morbidity and reduction in quality of life. Botulinum toxin (BT) is an established treatment for anal fissures despite there being no consensus on the optimal dosage or injection location for sustained healing of the fissure. Methodology: A systematic review was conducted according to PRISMA guidelines using the Medline, EMBASE, and CENTRAL databases. Randomized controlled trials with documented follow-up for at least 24 weeks without any confounding anal pathologies were included. The primary outcome was healing of the anal fissure with recurrences classified as treatment failures. Secondary outcomes were adverse effects of fecal or flatus incontinence. Botox dose was classified as Low dose (LD, 20-39 units) or High Dose (HD, 40-50 units). Results: Nineteen randomized controlled trials were included for analysis with 778 participants (51% F) with a mean age of 40.9 years all receiving BT injections into the internal anal sphincter. Mean follow-up post-treatment was 92.6 weeks. HD injected into the midline had the highest healing rate at 94% (p < 0.001) but carried the highest incontinence rate at 19%. LD into the lateral or midline of the internal anal produced similar rates of healing (68%, 64%) and incontinence rates (5%, 6.9%). Conclusion: Injections of 20-39U of BT into either the midline or lateral aspect of the internal anal sphincter produces sustained healing of anal fissures in > 60% of patients with minimal incontinence rates. Higher doses are associated with healing only if injected into the midline and carry a substantial, albeit temporary, incontinence risk.