Introduction: The purpose of the study is to explore the effect of 12 weeks of plyometrics, speed, and agility training on physical fitness variables in sub-elite Kho-Kho players. The lack of evidence on the effect of a plyometric intervention on the sport-specific skills of Kho-Kho players motivated this study to understand the importance of training in preventing or minimizing sport-related injury in Kho-Kho players. Methods: Forty male sub-elite Kho-Kho players, aged 18-25, with more than 3 years of experience were selected. The sample size was estimated based on literature using speed as the primary variable, as it is crucial for optimal performance in Kho-Kho players it was estimated as 20 in each group. Players were assigned either group through sequentially numbered, sealed, opaque envelopes, using computer-generated randomization. Physical fitness variables were assessed at baseline and post-intervention, including agility, power, muscular endurance, flexibility, speed, cardiovascular endurance, and balance. Outcome measures used were the Illinois agility test, Sargent jump test, one-minute squat test, plank test, modified sit and reach, 40-yard dash test, Yoyo intermittent recovery level 1 test, and lower quadrant Y balance test. Intergroup comparisons were performed using an independent t-test and intragroup comparisons using a paired t-test. (Level of significance ≤0.05). Results: The experimental group demonstrated the highest improvement in cardiovascular endurance (57.84%), followed by lower limb endurance (30.01%), explosive power (28.83%), speed (13.49%), agility (13.12%), and dynamic balance (9.09%) whereas the control group showed improvement in lower limb endurance (8.4%), followed by speed (4.01%) and dynamic balance (2.2%), with no significant difference observed in agility, power, cardiovascular endurance, and core endurance (p>0.05) Conclusion: Plyometric training integrated with conventional Kho-Kho training improves the sports-specific physical fitness of sub-elite Kho-Kho players. Implications: The study suggests that plyometric training with conventional Kho-Kho training improves sports-specific physical fitness, enhances athlete performance, and facilitates career development.