Rhipicephalus microplus is the tick species that causes the most problems worldwide, particularly affecting cattle farms. It represents a huge economic loss, impacting both animal health and farm profitability. Present in Réunion Island for several decades and imported with its hosts from Asia, it is now found throughout the island. Since the 1960s, this tick has been controlled largely through the use of acaricides. Today, farmers are highlighting the ineffectiveness of these treatments and pointing to the emergence of resistance in this species on Réunion farms. The aim of our study is to highlight 1) the levels of resistance in tick populations on the farms participating in the study and 2) the mechanism responsible for this resistance. First, we conducted resistance tests such as Larval Tarsal test (LTT) and then Larval Packet Test (LPT). Next, molecular biology tools such as PCR enabled us to explore the presence of a resistance gene associated with deltamethrin resistance. Bioassays enabled us to identify tick populations that were highly resistant to deltamethrin, which is widely used in Réunion Island. Our findings have provided a better understanding of the mechanisms at work in the resistance phenomenon of R. microplus and have guided farmers’ practices towards better tick management in relation to the resistance of local tick populations.