Background: The injuries sustained in football most frequently occur to the lower extremity, with ankle and knee being the most affected joint segments. Many studies have defined ankle sprain as one of the most common sports injuries with an annual incidence rate of 15% to 45%, however, there’s a lack of studies in football regional leagues. Risk factors like anthropometric measures, field position, dominant limb, previous injury have been described as being associated with ankle injuries in the elite sports context. The primary aim of this study was to determine prevalence rates of self-reported ankle instability in regional leagues. A secondary aim was to explore the presence of extrinsic factors affecting ankle instability in these football players. Methods: The heads of all clubs (n = 66) from Coimbra and Aveiro (Portugal) regional male senior football divisions during season 2016/2017 were contacted by e-mail or telephone to enter the study, and 58 gave their permission to include their athletes. Then, all players from each of these 58 teams were invited to enter the study (n = 1044) and a total of 589 athletes participated. Demographic and anthropometric data were recorded concerning age, height, weight, years of training, mean number of training hours, position in camp along with specific questions related to an ankle injury. All participants were asked about their previous history of injury, reinjury, feeling of giving-away and where also asked to fill the Cumberland Ankle Instability Tool. Results: A total of 290 (49.2%) athletes reported a history of at least one previous sprain and, from these, 170 (58.6%) repeated the injury and 112 (19%) reported sensation of “giving-away”. A significant association was found between injury, recurrence and subjective feeling of giving away (p < 0.05). The player field’s position was found to be associated with ankle instability, with defenders and forwards having higher rates of self-reported instability. No other significant associations were found. Conclusi