This study provides the first detailed information on the reproductive biology of the lesser guitarfish Zapteryx brevirostris in the south-western Atlantic Ocean between latitudes 34° and 42° S. A total of 320 males (196–647 mm total length, LT) and 336 females (200–640 mm LT) were analysed. No significant differences were observed in size at 50% maturity between males (499 mm LT) and females (506 mm LT). Three seasonal patterns were related to the testes mass, the development of spermatocysts and the presence of spermatozoa in genital ducts. Females showed a 3 year reproductive cycle, with 2 years of ovarian maturation and 1 year of gestation, including a mean annual fecundity of one to two embryos. Synchronicity of both ovulating females and males with sperm in genital ducts indicated that ovulation and hence mating occurs during winter. Birth was assumed to occur during winter, but neither ovulation nor mating is simultaneous with this reproductive event. Despite its low economic value, a decline in abundance in the region, together with a low reproductive efficiency, indicates that the Z. brevirostris population should be monitored closely.