Using the newly constructed Questionnaire on the incidence of pain and knee injuries in football, on a sample of 39 senior football players who participate in Croatian first league competitions, good metric characteristics of this measuring instrument were determined. The high reliability coefficient and the good sensitivity of the scale of the Questionnaire on the incidence of knee joint pain in football indicate that its application in further research is possible. Significant differences in the incidence of knee pain by gender were found in three of the ten variables used in this study. No significant difference according to the game line was found in any of the variables used using a ANOVA. A total of 63.16% of female football players reported the absence of a knee injury, while 55% of first-league football players reported the presence of a knee joint injury, indicating a higher presence of injuries in males. The most common knee injury refers to meniscus and anterior cruciate ligament injuries and is quantitatively represented with a relative value of 50.00%. The scientific contribution of this paper is reflected in the construction and validation of a new measuring instrument for determining the incidence of knee pain and injuries in football. Future research guidelines indicate the need to increase the number of respondents as well as the need to modify the questionnaire itself by increasing the number of particles and variables.