In a recent study, myotonometry revealed adverse alterations in the mechanical properties of ankle periarticular muscles in soldiers reporting previous lateral ankle sprain (LAS) incidents. This knowledge, if confirmed in athletes with developed chronic ankle instability (CAI), may have important diagnostic and therapeutic implications in sports medicine and rehabilitation. Consequently, the purpose of the study was to explore mechanical properties of the peroneus longus (PL), tibialis anterior (TA), and the lateral and medial gastrocnemius (LG and MG, respectively) muscles in athletes suffering CAI. Fourteen adult male athletes with developed CAI (International Ankle Consortium selection criteria) and 15 healthy male athletes without any LAS incidence as the control group (CO) participated in the study. The CAI and CO groups were characterized by similar anthropometric parameters and weekly training volume. In both the groups of athletes, we performed resting state (controlled with electromyographic recordings) myotonometric measurements (using a MyotonPRO® device) in the PL, TA, LG and MG muscles and calculated the following five myotonometric parameters: frequency (F-MYO), stiffness (S-MYO), decrement (d-MYO), relaxation time (R-MYO) and creep (C-MYO). Athletes from the CAI group (compared to the CO group) exhibited significantly higher values of F-MYO and S-MYO as well as lower values of R-MYO and C-MYO in the PL and TA muscles. No significant inter-group differences were found in the remaining myotonometric parameters. Using myotonometry, our study is the first to reveal that athletes with developed CAI are characterized by heightened tone and stiffness with concurrent lowered elasticity of the PL and TA muscles.