Golfers place alignment marks on their balls in the belief that this improves the ability to align and hole more putts. The purpose of this study was to determine the effect of using an alignment mark on a golf ball on the number of putts holed. Twenty-nine amateur golfers (handicap 7.6 ± 4.06) performed 10 straight putts from a distance of 1.7m to ascertain putting ability. Based on putting ability they then performed 30 putts from a distance of 1.7m, 2.3m or 2.6m, with better putters putting further from the hole. Fifteen of the putts were performed using a ball with an alignment mark (AM) and 15 with no alignment mark (NAM). The order of putts with each ball was randomized. On average participants holed 17.1 ± 3.9 putts out of the 30 putts completed using both balls. The number of putts holed at each of the three distances from the hole did not differ (*p* = .60). More putts were holed with AM compared with NAM at a distance of 2.6m (AM 9.7 ± 2.4 putts, NAM 7.6 ± 2.8, *p* \< .01), but not at the shorter distances of 1.7m (*p* = .67) and 2.3m (*p* = .39). The use of an alignment mark on a golf ball improves putting success for straight putts from a distance of 2.6m.