Amitraz is a broad-spectrum insecticide for the control of aphids, psyllids, ticks and mites. Current evidence suggests that ticks and phytophagous mites have developed strong resistance to amitraz. Previous studies have shown that the I61F mutation in the β-adrenergic octopamine receptor is related to amitraz resistance in ticks, but the mutation was not found in Panonychus citri. We therefore used whole genome sequencing and bulked segregant analysis to identify the mechanism by which P. citri is resistant to amitraz. High-quality assembly of the whole P. citri genome was completed, resulting in a genome of approximately 83.97 Mb and a contig N50 of approximately 1.81 Mb. Gene structure predictions revealed 11,577 genes, of which 10,940 genes were annotated. Trait-associated regions in the genome were mapped with bulked segregant analysis and 38 candidate SNPs were obtained, of which T752C had the strongest correlation with the resistant trait, located at the 5’ untranslated region (UTR) of the β-2R adrenergic-like octopamine receptor gene. The mutation resulted in the creation of a short hairpin loop structure in mRNA and gene expression was down-regulated by more than 50% in the amitraz-resistant strain. Validation of the T752C mutation in field populations of P. citri found that the correlation between the resistance ratio and the base mutation was 94.40%. Our results suggest that this 5’ UTR mutation of β-2R octopamine receptor gene confers amitraz resistance in P. citri and different species may share different mechanisms of resitance to amitraz.