Background: Chronic anal fissure is a common disease of the anoderm. Botulinum toxin injection has recently been recommended. However, the injection sites are still debatable. Aims: This study aimed to assess the site-dependent efficacy of Botulinum toxin injection for chronic anal fissure healing. Methods: Randomized clinical trial was performed, 80 patients were enrolled in two semi groups according to the site of botulinum toxin injection. Lateral sites of internal sphincter muscle were used in group 1, anterior and posterior regions were used in group 2 in lithotomy position. In this prospective study, the effect of injection site preference on the early complications (pain, infection, hematoma, incontinence) and late results (clinical fissure healing) of chronic anal fissure treatment were assessed. Results: There was no statistical significant difference between the number of patients who achieved complete healing at the end of the 12th week. Postoperative pain scores were significantly lower in group 2 at the end of 1st week and they stayed lower at the end of the 2nd week. Thrombosed haemorrhoids were encountered significantly lower in group 2. Conclusions: Even though there was no statistical difference in the long term healing rate between groups 1 and 2, choosing 6 and 12 o'clock alignments of internal sphincter muscle for botox injection is more advantageous than 3 and 9 o'clock alignments in terms of better postoperative pain and lower complication rate in the early period.