A study was conducted to assess the relative effect of CaCl2 on the breast and thigh muscles of spent White Leghorn layers. A total of 96 muscle samples obtained from 24 spent hens were used to study the effect of CaCl2 injection on physico-chemical properties of breast and thigh muscles. Physico-chemical parameters like pH, water holding capacity, protein extractability, shear force value and myofibrillar fragmentation indices of breast and thigh muscles significantly (P≤0.01) varied with injection of CaCl2 with 24 hr of ageing. Significant (P≤0.01) difference was observed for L* and a* values between control breast or thigh and corresponding treated muscles, no significant (P≥0.05) difference was observed for b* values between control and treated muscles. However, the color differences (ΔE) values among all groups were over the just-noticeable difference threshold. Discriminant function analysis and Mahalanobis distance (D2) analysis of physico-chemical parameters demonstrated that the effect of CaCl2 is comparatively greater in thigh muscle than breast muscle with an ageing period of 24 hr.