Isolation of non-tuberculous mycobacteria (NTM) from the sputum of patients with cystic fibrosis (CF) is challenging due to overgrowth by rapidly-growing species that colonise the lungs of patients with CF. Extended incubation ofBurkholderia cepaciaselective agar (BCSA) has been recommended as an expedient culture method for isolation of rapidly-growing NTM in this setting. The aim of this study was to assess the efficacy of five selective media designed for the isolation ofBurkholderia cepaciacomplex (BCC) along with two media designed for the isolation of mycobacteria (RGM medium and Middlebrook 7H11 agar) for their ability to isolate NTM. All seven media were challenged with 147 isolates of rapidly-growing mycobacteria and 185 isolates belonging to other species. RGM medium was then compared with the most selective brand of BCSA for the isolation of NTM from 224 sputum samples from patients with CF. Different agars designed for isolation ofB. cepaciacomplex varied considerably in their inhibition of other bacteria and fungi. RGM medium supported the growth of all isolates of mycobacteria and was more selective than any other medium. NTM were recovered from 17 of 224 sputum samples using RGM medium compared with only seven samples using the most selective brand of BCSA (P= 0.023). RGM medium offers a superior option to other selective agars for isolation of rapidly-growing mycobacteria from the sputum of patients with CF. Furthermore, the convenience of using RGM medium enables routine screening for rapidly-growing NTM in all sputum samples submitted by patients with CF.