Grapefruit cultivars are highly sensitive to CTV infections and in order to increase their productive lifespan, the Southern African citrus industry makes use of cross-protection. However, the breakdown of cross-protection is commonly observed in commercial grapefruit plantings. In order to determine which genotypes of CTV are associated with commercial Citrus x paradisi (Macfad.) cv. 'Star Ruby' in Southern Africa, 192 samples, pre-immunised with the GFMS 12 population, were collected from the grapefruit production areas of Hoedspruit, Malelane, Swaziland, Northern Cape, Sundays River Valley and Nkwalini Valley and six samples from non-pre-immunised plants in Letsitele as well as three samples from greenhouse maintained plants harbouring populations derived from the original GFMS 12 source. The p33 gene was amplified with direct Sanger sequencing performed on the resulting amplicons. A subset of 92 samples randomly selected and p33 gene amplicons subjected to Illumina MiSeq amplicon sequencing. High levels of CTV diversity were observed between and within orchards. Most populations were made up of a dominant component with several minor sequence types. Resistance Breaking (RB) sequences were most numerous, especially in recently planted orchards and present within all of the populations. The Kpg3/SP/T3 group appeared to be the second most prevalent, with increased prevalence in older orchards. Sequences mapping to HA 16–5, VT, AT-1, T36, Taiwan-Pum/M/T5 and T30, were represented sporadically within numerous collection sites.