Initial genetic studies in Drosophila suggested that several members of the Rho subfamily (RhoA, Rac1 and Cdc42) are involved in planar cell polarity (PCP) establishment. However, analyses of Rac1, Rac2 and Mtl loss-of-function (LOF) mutants have argued against their role in this process. Here, we investigate in detail the role of the Rho GTPases Mtl, Cdc42, Rac1 and Rac2 in PCP generation. These functional analyses were performed by overexpressing Mtl in eyes and wings, by performing genetic interaction assays and by using a combination of triple and quadruple mutant LOF clones. We found that Mtl overexpression caused PCP phenotypes and that it interacted genetically with other Rho GTPases, such as Rac1 and Cdc42 as well as with several PCP genes, such as stbm, pk and aos. However, Mtl was not found to interact with Rac2, RhoA and other members of the Fz/PCP pathway. Triple mutant clones of Rac1, Rac2 and Mtl were found to exhibit mild PCP defects which were enhanced by reduction of Cdc42 function with a hypomorphic Cdc42 allele. Taken together, these and previous results suggest that Rho GTPases may have partially overlapping functions during PCP generation. Alternatively, it is also possible that the mild PCP phenotypes observed could indicate that they are required at low levels in that process. However, since not all of them function upstream of a JNK cassette, we propose that they may act in at least two parallel pathways.