Sugarcane is of important economic value for producing sugar and bioethanol. Tripidium arundinaceum (old name: Erianthus arundinaceum) is an intergeneric wild species of sugarcane that has desirable resistance traits for improving sugarcane varieties. However, the scarcity of chromosome markers has hindered the cytogenetic study of T. arundinaceum. Here we applied maize chromosome painting probes (MCPs) to identify chromosomes in sorghum and T. arundinaceum using a repeated fluorescence in situ hybridization (FISH) system. Sequential FISH revealed that these MCPs can be used as reliable chromosome markers for T. arundinaceum, even though T. arundinaceum has diverged from maize over 18 MYs (million years). Using these MCPs, we identified T. arundinaceum chromosomes based on their sequence similarity compared to sorghum and labeled them 1 through 10. Then, the karyotype of T. arundinaceum was established by multiple oligo-FISH. Furthermore, FISH results revealed that 5S rDNA and 35S rDNA are localized on chromosomes 5 and 6, respectively, in T. arundinaceum. Altogether, these results represent an essential step for further cytogenetic research of T. arundinaceum in sugarcane breeding.