Rice has been one of the most important plant resources both in modern and ancient times. Research on the origin and domestication of rice is an important topic in archaeobotany; phytolith analysis has played a crucial role improving understanding of these issues. In this study, we collected 30 specimens including 28 Oryza (rice) species, 1 Leersia species, and 1 Zizania species to perform in situ observation of phytolith in different parts of the spikelet. In situ observation revealed that double-peaked phytoliths were derived from two nearby epidermal long cells and located various phytolith morphotypes in different parts of the spikelet. We found that the double-peaked phytoliths, Rondel tridentate and Rondel reniform distinguished specimens at genus level; the morphology and morphometry of double-peaked phytoliths distinguished rice specimens from different genomes; and higher amount of Fusiform echinate in the rachilla might reflect the non-shattering phenotype of rice within the AA genome. Our study adds detailed information regarding phytolith morphology and distribution, and provides new tools and insights for the future archaeobotanical research.