Perennial legumes have potential to increase pasture productivity in the high rainfall zone (600–850 mm) of south-eastern Australia through their ability to use summer rainfall and fix nitrogen (N2). Various perennial legumes are being evaluated for this environment; however, little information exists on legume–rhizobia cross-host compatibility and its consequences for biological N2 fixation. This is especially important when legumes are sown into fields with a background of competitive rhizobia such as WSM1325 or sown as a pasture mix with different host–symbiont pairs. We studied the effectiveness and cross-host compatibility of five commercial rhizobial strains for a range of pasture legumes (nine species, 18 cultivars) under controlled environment conditions, and further evaluated nodule occupancy and competitiveness of a newly established pasture (13 species, 20 cultivars) in the field, by determining nodulation and production (biomass and N2 fixation). Three of the commercial inoculant strains formed root nodules with multiple legume species; commonly however, less N2 was fixed in cases where the inoculant was not the recommended strain for the legume species. Within a legume species, cultivars could differ in their ability to form effective root nodules with multiple rhizobial strains. White clover cvv. Trophy, Haifa and Storm, strawberry clover cv. Palestine, and Talish clover cv. Permatas formed effective nodules with both TA1 and WSM1325 rhizobial strains. White clover cultivars that could not form an effective symbiosis with the common background strain WSM1325 fixed less N2. The white clover × Caucasian clover hybrid formed effective symbiosis with strain TA1 but not with other commercial strains. Some species such as birdsfoot trefoil, Talish clover, sulfur clover and tetraploid Caucasian clover formed ineffective symbiosis in the field. Until resolved, this will likely inhibit their further development as pasture plants for similar permanent pasture environments.