Objectives To investigate β-alanine supplementation use and level of knowledge amongst professional footballers. Design Cross-sectional survey of Australian professional football players. Methods Questionnaires assessing β-alanine supplementation behaviours, level of knowledge and sources of information were completed by professional rugby union (RU) (n = 87), rugby league (RL) (n = 180) and Australian Rules Football (ARF) (n = 303) players. Results Approximately 61% of athletes reported β-alanine use, however use by ARF football players (44%) was lower than that of RU (80%) and RL players (80%). The majority of respondents were not using β-alanine in accordance with recommendations. Only 35% of the participants were able to correctly identify the potential benefits of β-alanine supplementation. The main information sources that influenced players’ decision to use β-alanine were strength and conditioning coach (71%) and dietitian (52%). Forty-eight per cent of athletes never read labels prior to supplementing and only 11% completed their own research on β-alanine. Compared to RL and ARF players, RU players had both a greater knowledge of β-alanine supplementation and better supplementation practices. Conclusions Despite over half the surveyed professional footballers using β-alanine, the majority of athletes used β-alanine in a manner inconsistent with recommendations. A better understanding of the environment and culture within professional football codes is required before supplement use becomes consistent with evidence based supplement recommendations.