In this experiment we investigated the hypothesis that heterogeneity of plant structures presents disparity in niches available for colonisation by the rumen microbiota resulting in differential colonisation. Fresh perennial ryegrass (PRG) stem and leaves were incubated in the presence of rumen bacteria under rumen-like conditions with incubations harvested at many time intervals up to 24 h. In vitro dry matter degradability (IVDMD) of stem material was lower than that of leaves at all harvesting times. Denaturing Gradient Gel Electrophoresis (DGGE) derived dendrograms, canonical analysis of principal coordinates (CAP) and PERMANOVA demonstrated that diversity of bacteria attached to PRG stem and leaf material was different at all harvesting times although QPCR data showed similar quantities of bacterial 16S rDNA on stem and leaf material at all harvesting times. Conversely, bacterial diversity on abaxial and adaxial leaf surfaces was similar, but 16S rDNA quantity differed with more 16S rDNA on the adaxial surface at all harvesting times. Image analysis of low temperature scanning electron microscopy (LTSEM) pictures confirmed that biofilm coverage on the adaxial surface was greater than the abaxial surface. We demonstrate that differing plant parts can affect attached bacterial diversity and/or 16S rDNA quantity present. This result is consistent with concepts of niche specialisation by the rumen microbiota. This observation is particularly relevant to understanding rumen plant-microbe interactions which is necessary for development of novel strategies for improving ruminant nutrient use efficiency.