Cotton leaf curl disease (CLCuD) is the major biotic limitation to cotton production in Pakistan and northwestern India. The disease is caused by various distinct viruses of the genus Begomovirus (family Geminiviridae) in association with a disease-specific betasatellite - cotton leaf curl Multan betasatellite (CLCuMB). Since 2001, when resistance to CLCuD in cotton was broken, only one virus was consistently identified in cotton exhibiting CLCuD symptoms in Punjab province (Pakistan) - cotton leaf curl Burewala virus (CLCuBuV). An analysis of all CLCuBuV isolates available in the databases showed these to represent only a single strain, based on currently applicable criteria. Virus and betasatellite clones were obtained from a leaf sample (isolate C49) collected from a CLCuD symptomatic cotton plant in Layyah district, Punjab province, in 2012. Analysis of the sequence of the betasatellite showed this to be an isolate of CLCuMB containing the recombinant fragment typical of this satellite post-resistance-breaking. The virus was shown it to be an isolate of CLCuBuV but to be distinct from all previously characterised isolates and to represent a distinct strain. In common with previous CLCuBuV isolates, the virus from C49 is a recombinant containing sequences derived from viruses of two species that were prevalent in cotton pre-resistance-breaking but with distinct recombination break sites. As was the case with the earlier CLCuBuV, the newly identified strain of CLCuBuV lacks an intact transcriptional activator protein.