The burrowing nematode, Radopholus similis, is the most important nematode pest of banana and plantain worldwide, causing severe yield losses in commercial and local consumption cultivars. Chemical control is currently the most-used method to manage the nematodes, although nematicides are dangerous, toxic and expensive. Therefore, nematode control through genetic improvement is widely encouraged. Protocols have been set up to detect sources of resistance in the existing Musa germplasm, but the screening under greenhouse and field conditions is laborious and time consuming. In vitro screening could facilitate this process by picking up sources of resistance at an early phase. In this study, a procedure for in vitro screening for the resistance to R. similis was set up by determining the optimal incubation time and the optimal nematode inoculum. In a second step, the optimized procedure was confirmed by screening a selection of Musa varieties with a known host plant response. Except for ‘Yangambi Km5’, their host response under in vitro conditions corresponded to their host response under greenhouse or field conditions. The susceptible status of ‘Grande Naine’, ‘Gros Michel’ and ‘Cachaco’ was confirmed, as well as the resistant status of ‘Pisang Jari Buaya’ and ‘SH-3142’.