Positron annihilation lifetime (PAL) technique is applied on natural rubber to determine the parameters of nano free volumes such as size, concentration and distribution as a function of temperature. In agreement with the Differential Scanning Calorimeter (DSC) measurements and X-ray diffraction (XRD) measurements, the results showed that the (PAL) technique is sensitive to probe the glass transition temperature (Tg) and its ability to determine the melting transition temperature (Tm). Above Tg, the isothermal measurements showed an intermediate transition point before the melting point. Moreover, the ortho-positronium (o-Ps) was able to probe the variation of free volumes in the amorphous domains with the change of temperature. The calculated expansion coefficient of free volume indicates that o-Ps is selectively probing free volume sites in amorphous domains.