The present communication deals with the detection and characterization of deltamethrin resistance in tick populations using biological (larval packet test), biochemical (esterase enzyme assay) and molecular assays. Ticks were collected from cattle farms of Korutla, Telangana (KOR), Mehboob Nagar, Telangana (MBN), Nagpur, Maharashtra (NAG), Parbani, Maharashtra (PBN), Madhavaram, Tamil Nadu (MAD), Cuddalore, Tamil Nadu (CUD), Sakhleshpur, Karnataka (SAK) and Buvenduvella, Karnataka (BUV). Out of eight field isolates, seven were identified as Rhipicephalus (Boophilus) microplus while one isolate (CUD) was identified as R. (B.) annulatus. The LC50 values and resistance factors (RF) of field isolates were assessed by larval packet test (LPT). RF values of two isolates viz., Korutla and Parbhani (KOR, PAR) were close to that of reference susceptible isolate. R. (B.) microplus isolate from Nagpur (NAG) and Sakleshpur (SAK) revealed slightly higher RF values (6.42 and 4.51). They revealed slightly elevated esterase enzyme activity too. Other isolates did not reveal higher values for RF or esterase activity. Previously identified mutations conferring synthetic pyrethroid resistance in R. (B.) microplus populations were analysed by sequencing the mutation flanking regions of the carboxyl esterase and the sodium channel genes (domain III S6 and domain II S4-5 linker region). However, these point mutations were not detected in the field isolates. The results of the present study revealed that low levels of synthetic pyrethroid resistance had developed in field populations of ticks of southern India.