Miscanthus sinensis Anderss. (Poaceae) has desirable traits for a dedicated biomass crop. An important breeding goal for M. sinensis is to develop F1 hybrid cultivars. A clear understanding of its reproductive mode will help to identify effective breeding strategies toward that goal. We performed >1000 semi-in-vivo reciprocal crosses and self-pollinations to determine pollen–pistil compatibility responses. Self-pollination showed a self-incompatibility (SI) response typical for grasses, indicating that SI is operative in M. sinensis. The majority of self-pollen produced short tubes that terminated at the stigmatic surface of mature pistils, but some self-pollen tubes entered into the transmitting tract. The developmental stage of pistils affects pollen–pistil interactions, as younger pistils allowed significantly more self-pollen to enter the transmitting tract, with some even reaching the ovule. Cross pollinations among progeny of reciprocal crosses between the cultivar Gross Fontaine and the cultivar Undine showed four classes of compatibility responses with 0, 50, 75, and 100% compatible pollen and exhibited differences in reciprocal compatibility for certain crosses. Taken together, our results showed that SI in M. sinensis is gametophytic and is likely controlled by a multiallelic, two-locus S and Z system, similar to those reported for other grasses. The findings from this study will facilitate the identification and isolation of genes related to SI and ultimately F1 hybrid production in M. sinensis.