Spontaneously expectorated sputum is traditionally used as the sampling method for the investigation of lower airway infections. Whilst guidelines exist for the handling of these samples for culture-based diagnostic microbiology, there is no comparable consensus on their handling prior to culture-independent analysis. The increasing incorporation of culture-independent approaches in diagnostic microbiology means it is of critical importance to assess potential biases. The aim of this study was to assess the impact of delayed freezing on culture-independent microbiological analyses, and to identify acceptable parameters for sample handling. Sputum samples from eight adult cystic fibrosis (CF) patients were collected and aliquoted into sterile Bijou bottles. Aliquots were stored at room temperature before freezing at -80°C for increasing intervals up to 72 hour period. Samples were treated with propidium monoazide, to distinguish live from dead cells, prior to DNA extraction, and 16S rRNA gene pyrosequencing was used to characterise the bacterial composition. Substantial variation was observed in samples with high diversity bacterial communities over time, whereas low diversity communities dominated by recognised CF pathogens varied little regardless of time to freezing. Partitioning into common and rare species demonstrated that the rare species drove changes in similarity. The percentage abundance of anaerobes over the study significantly decreased after 12 hours at room temperature (P=0.008). Failure to stabilise samples at -80°C within 12 hours of collection results in significant changes in the detected community composition.