Background: Culture-independent sequencing methods are increasingly used to investigate the microbiota associated with human mucosal surfaces, including sites that have low bacterial load in healthy individuals (e.g. the lungs). Standard microbiota methods developed for analysis of high bacterial load specimens (e.g. stool) may require modification when bacterial load is low, as background contamination derived from sterile laboratory reagents and kits can dominate sequence data when few bacteria are present. Main body: Bacterial load in respiratory specimens may vary depending on the specimen type, specimen volume, the anatomic site sampled and clinical parameters. This review discusses methodological issues inherent to analysis of low bacterial load specimens and recommends strategies for successful respiratory microbiota studies. The range of methods currently used to process DNA from low bacterial load specimens, and the strategies used to identify and exclude background contamination are also discussed. Conclusion: Microbiota studies that include low bacterial load specimens require additional tests to ensure that background contamination does not bias the results or interpretation. Several methods are currently used to analyse the microbiota in low bacterial load respiratory specimens; however, there is scant literature comparing the effectiveness and biases of different methods. Further research is needed to define optimal methods for analysing the microbiota in low bacterial load specimens.