Tire wear particles (TWPs) are generated by friction between the road and the tire. TWPs are one of the major microplastics found in environmental samples, such as road dust, particulate matter (PM), and sediment. TWP contents in environmental samples are generally analyzed using the pyrolysis technique. Tire tread compounds of heavy vehicles are usually composed of natural rubber (NR). Isoprene and dipentene are the principal pyrolysis products of NR, and dipentene is employed as the key marker for the determination of the TWP contents. In this study, an NR abrasion specimen was thermally aged, and an abrasion test was performed to obtain the wear particles. The influence of the wear particle size and thermal aging on the pyrolysis behavior of NR was investigated. The isoprene/dipentene ratio exponentially increased as the wear particle size decreased, and it was also increased by the thermal aging of the abrasion specimen. The increased isoprene/dipentene ratio by thermal aging was explained by increasing the crosslink density. Using the relationship between the wear particle size and the isoprene/dipentene ratio, it is possible to estimate the isoprene/dipentene ratio for very small TWP such as PM. The experimental results concluded that the wear particle size and thermal aging affect the formation of the key pyrogenic products, and the influencing factors should be considered for the quantification of TWP contents in the environmental samples.