The archaeological site of Tlatoani at Tlayacapan is located in the Mexican Highlands, in the present-day state of Morelos. The site is an extant settlement located at the top of the Tepoztlan mountain range, and has been occupied since the Late Preclassic period (AD 150–500). At the height of its occupation in the Epiclassic and Early Postclassic periods (AD 600–1150), Tlayacapan was situated on the top of the hill. The radiocarbon investigations reported herein revealed some further distinct findings, although no clear absolute chronology was demonstrated. A dog skull was found inside the oldest foundation stage, and dated between cal AD 646 and 765, the middle of the Epiclassic period. Human remains found in the first grave belonged to three individuals. A male skeleton was dated to AD 1158–1227. Fragments of an incomplete skeleton of a child and an incomplete skeleton of a second male were placed on top of the first male skeleton and were dated in the range AD 1030–1156. A fourth skeleton found nearby in the second grave gave a similar date of AD 1164–1253. These burials were in accordance with the Middle America cosmovisional system, where bodies were buried beneath the household space. It is evident from the 14C dates of the skeletons that the burial sites beneath the household space had been reused by exhuming and reburying skeletons that had been previously buried there. A comparison of dates on fractions of collagen and bioapatite of the same bones was possible. Two of the samples were in good agreement between these fractions, whereas the other three samples are close but just outside the 2σ range.