Insecticide resistance is a serious threat to vector control programs worldwide. Therefore, it is crucial to monitor the development of resistance in vector populations. Rhipicephalus linnaei (Audouin, 1826) is a cosmopolitan tick and a vector of medically important pathogens. We conducted a comprehensive investigation of permethrin resistance in larvae of Rh. linnaei populations across Thailand by comparing phenotypic resistance with tick genotypes, focusing on mutations in Domain II and III of the voltage-gated sodium channel gene. Results showed that larvae obtained from engorged female tick populations in Thailand have developed resistance to permethrin, with levels varying by location. Resistance ratios ranged from 1 to 56 when compared to the least susceptible local population. Genotyping identified mutations at positions 190 (c.190C >
A and c.190C >
G) and 2134 (c.2134T >
C) in Domain II and Domain III, respectively, which are correlated with phenotypic resistance. We identified new alleles c.190CG and c.190AG in highly resistant populations from Phasichareon, Bangkok, and Chonburi provinces. This study provides the first evidence, to our knowledge, of permethrin resistance in Rh. linnaei ticks in Thailand. Elevated levels of permethrin resistance in Rh. linnaei populations across Thailand indicate that veterinarians and farmers should consider tick control products with alternative modes of action.