Background: Children with cystic fibrosis (CF) can develop life-threatening infections of Mycobacterium abscessus. These present a significant clinical challenge, particularly when the strains involved are resistant to antibiotics. Recent evidence of within-patient subclones of M. abscessus in adults with CF suggests the possibility that within-patient diversity may be relevant for the treatment of pediatric CF patients. Methods: We performed whole genome sequencing (WGS) on 32 isolates of M. abscessus from multiple body sites for two patients with CF undergoing treatment at Great Ormond Street Hospital, UK, in 2015. Results: We found evidence of extensive diversity within patients over time. Clustering analysis of single nucleotide variants (SNVs) revealed that each patient harboured multiple subpopulations, which were differentially abundant between sputum, lung samples, chest wounds, and pleural fluid. Sputum isolates did not reflect overall within-patient diversity, including failing to detect subclones with mutations previously associated with macrolide resistance (rrl 2058/2059). Some variants were present at intermediate frequencies before lung transplant. The time of transplant coincided with extensive variation, suggesting that this event is particularly disruptive for the microbial community, but transplant did not clear the M. abscessus infection and both patients died as a result of this infection. Conclusions: Isolates of M. abscessus from sputum do not always reflect the entire diversity present within the patient, which can include subclones with differing AMR profiles. Awareness of this phenotypic variability, with sampling of multiple body sites in conjunction with WGS, may be necessary to ensure the best treatment for this vulnerable patient group.