Cladosporium is widespread in Heating, Ventilation and Air Conditioning (HVAC). Some species in which are responsible for the majority of fungal infections or allergy in humans. To assess the exposure risks of Cladosporium in HVAC to human health, the colony distribution, population constituent and pathogenic species identification of Cladosporium were investigated in current study. We selected two cities, Beijing and Nanjing, representing the North and South of China respectively, for the study. Three kinds of public places including hospitals, hotels and super markets were selected and 108 air samles from 18 sampling sites were collected from air supply outlets of HVAC in summer, 2012. The collecting plates were incubated at 28°C for 2-3 weeks. Purified fungal colonies were identified at the genus and species level based on morphological and DNA sequences. Internal transcribed spacer ITS1 and ITS2 region, partial gene sequences of the translation elongation facter1-α gene (EF-1α) and the actin gene (ACT) were used for phylogenetic analysis. Among all genera detected, Cladosporium was identified to have the widest prevalence (92.3%) from HVAC, which was detected from 100 of 108 air samples. 9 species of Cladosporium were identified viz. C. breviramosum, C. cladoporioides, C. cucumerinum, C. gossypiicola, C. macrocarpum, C. perangustum, C. sp., C. sphaerospermum and C. tenuissimum. Among of them, C. cladosporioides had the highest concentration and frequency, considered as predominant species in HVAC. There were three species, C. cladoporioides, C. sphaerospermum and C. macrocarpum, distributed as opportunistic pathogen to human reported before. The total frequency of the three species was 76.29% of this genus. What’s more, C. cladoporioides and C. sphaerospermum were detected in both cities with relatively high concentration and frequency. The frequency and prevalence rate in the South were higher than those in the North, suggesting that HVAC in the South was more susceptable to Cladosporium contamination.