Background: In the treatment of chronic anal fissures (CAFs), surgical sphincterotomy is more commonly being replaced by chemical sphincterotomy. After the good results of our pilot study including 32 patients, we now describe the effect of botulinum toxin A (BT-A) in a consecutive series of 100 patients with isosorbide dinitrate (ISDN) ointment-resistant CAFs. Methods: From October 2002 until August 2005, 100 patients (52 males, 48 females) with a median age of 45 (20–79) years were treated with an injection of 40–100 IU BT-A (Dysport®, Ipsen, The Netherlands) in the internal anal sphincter. Results: After a median follow-up of 10 (4–38) months, 77 of the 100 CAFs (77%) were cured. 20 patients were given a second injection, 1 a third and 1 a fourth injection. In 11 patients a fissure recurred (14%). In 1 patient (1%) there was temporary incontinence due to flatus. Conclusion: With an early response rate of 77% and an overall success rate of 66%, BT-A injections appear to be effective in patients with ISDN ointment-resistant CAFs if initial non-responders are retreated. These results are in concordance with the results of our pilot study. It is a simple technique with little or no side effects, that does not compromise future treatments. Its place in the first-line treatment of CAFs should be investigated further.