Pollen from wind-pollinated plants is the predominant cause of pollinosis which corresponds mainly to allergic rhinitis and conjunctivitis. Bacterial communities associated to leaves and roots surface were analysed for many years with both culture-dependent and independent methods. However, microbiota of pollen grains was rarely investigated and never with a molecular fingerprint technique. The aim of this study was to perform a preliminary analysis of the pollen microbiota by applying PCR-TTGE method. Pollen samples were collected from various anemophilous trees genera (Cephalotaxus, Cupressus, Pinus, Platanus and Quercus) in Montpellier (France) in 2013. Thorough pollen preparation was essential to a successful recovery of DNA from pollen. Regarding the results, pollen microbiota was tree genus-dependent. In addition, intra-tree genus variations were also observed. The Gammaproteobacteria class was the most represented in pollen samples whatever the tree genus. Among this class, some bacteria were recognized as phytopathogens (Pseudomonas, Erwinia) but also opportunistic human pathogens (Pseudomonas, some enterobacteria). PCR-TTGE is a suitable tool to analyse the microbiota associated with pollen responsible for phytopathogenicity or affecting the human respiratory tract.