| Summary: | This study delves into the exploration of genetic diversity and phylogenetic relationships within <i>Camellia reticulata</i> cultivars, providing a vital reference for horticultural research on this hetero-hexaploid species. Utilizing available transcriptome data from <i>C. reticulata</i>, novel low-copy nuclear gene sequences were successfully identified. With tailored primer design, these genes were amplified and sequenced from 100 <i>C. reticulata</i> cultivars originating from Kunming, Chuxiong, Dali, and Tengchong in China. Five distinct low-copy nuclear gene sequences were found to collectively span 3481 bp, showcasing 71 polymorphic mutation sites (<i>P</i><sub>i</sub> = 0.0077) and 91 haplotypes (<i>H</i><sub>d</sub> = 0.9974). The genetic diversity among cultivars from the four provenances ranked Tengchong > Dali > Kunming > Chuxiong. AMOVA analysis revealed that 96.50% of the genetic variation exists within the provenances. Low genetic distance and differentiation (<i>F<sub>st</sub></i>= 0.0199) were observed among cultivars from the four provenances. Ninety-nine cultivars were clustered into four clades, corresponding to the three ancestors of <i>C. reticulata</i> (diploid <i>C. reticulata</i>, <i>C. pitardii</i>, and <i>C. saluenensis</i>). The novel low-copy nuclear gene sequence markers developed in this study provide an effective tool for analyzing genetic diversity, phylogenetic relationships, and origination of <i>C. reticulata</i> cultivars.
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