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Simple Summary: The number of thoracic vertebrae (TN) and lumbar vertebrae (LN) in Dezhou donkey population is different, which leads to the difference of meat production and skin yield, and it is regulated by a few genes. Nuclear receptor subfamily 6 group A member 1 (NR6A1) was found to be related to livestock vertebra development, but it is not reported in donkeys yet. In this study, seven single nucleotide variants (SNVs) were detected in the NR6A1 gene, and polymorphism information content (PIC) was moderate to high in the population. Then we analyzed the relationship between these SNVs and body size trait, carcass traits and the number of thoracolumbar vertebrae (TLN). We found that locus were associated with different traits, and the mutation effect was not completely consistent. The results suggested that these genetic variations in the NR6A1 gene may play an important role in regulating the development of thoracolumbar vertebrae of Dezhou donkey. This paper provides important preliminary work for the study of multi thoracolumbar vertebrae in Dezhou donkeys. The number of thoracolumbar vertebrae is a quantitative trait positively correlated with the economic traits of livestock. More thoracolumbar vertebrae individuals could genetically be used to improve the livestock population, as more thoracolumbar vertebrae means a longer carcass, which could bring more meat production. Nuclear receptor subfamily 6 group A member 1 (NR6A1) is considered a strong candidate gene for effecting the number of vertebrae in livestock. The purposes of this study are as follows: (a) Analyzing the effect of TLN variation on body size and carcass traits of Dezhou donkey; (b) Studying the distribution of seven single nucleotide variants (SNVs) in NR6A1 gene of Dezhou donkey; (c) Exploring the relationship between latent SNVs and TLN, the body size and carcass traits. We examined the thoracic and lumbar vertebrae number and seven SNVs in NR6A1 gene of 455 Dezhou donkeys, and analyzed the relationships between them. Five types of thoracolumbar combinations (T17L5 (individual with 17 thoracic and five lumbar vertebrae) 2.4%, T18L5 75.8%, T19L5 1.1%, T17L6 11.9%, and T18L6 8.8%) of Dezhou donkeys were detected in this study. For one thoracolumbar vertebra added, the body length of Dezhou donkey increases by 3 cm and the carcass weight increases by 6 kg. Seven SNVs (g.18093100G > T, g.18094587G > T, g.18106043G > T, g.18108764G > T, g.18110615T > G, g.18112000C > T and g.18114954T > G) of the NR6A1 gene were found to have a significant association with the TLN, body size and carcass traits of Dezhou donkey (p < 0.05), respectively. For instance, g.18114954C > T is significantly associated with lumber vertebrae number, the total number of thoracolumbar, and carcass weight, and individuals with TT genotype had significantly larger value than CC genotype (p < 0.05). Using these 7SNVs, 16 different haplotypes were estimated. Compared to Hap3Hap3, individuals homozygous for Hap2Hap2 showed significantly longer length in one thoracic spine (STL), the total thoracic vertebrae and one thoracolumbar spine. Our study will not only extend the understanding of genetic variation in the NR6A1 gene of Dezhou donkey, but also provide useful information for marker assisted selection in donkey breeding program. [ABSTRACT FROM AUTHOR]