After the introduction of the invasive cattle tick Rhipicephalus microplus in West Africa in the last decade, farmers encounter ticks resistance to the use of acaricides in different region in Benin. In order to evaluate the level of resistance, an in vitro study was performed on five samples of R. (Boophilus) microplus collected from five farms in four of the eight agro-ecological zone of Benin. The districts concerned with the study in the agro-ecological zone were Houeyogbe (Kpinnou), Zangnanado (Samiondji), Tchaourou (Okpara), Gogounou (Fana) and Bassila (Manigri). A toxicological test, the Larval Packet Test (LPT) was performed in the laboratory of Biotechnology Research Unit of the Animal Production and Health (URBPSA) at the Polytechnic School of University of Abomey-Calavi in Benin with the susceptible, Rhipicephalus geigyi strain from Hounde in Burkina Faso. Three (3) acaricides commonly used by farmers in Benin to control ticks were evaluated: alpha-cypermethrin, deltamethrin and amitraz. The results showed that the resistance ratio at 50% (RR50 95% CI) for the whole experiment varies from 1.96 to 338.5. Based on RR50 and RR90 values, only the population of Samiondji’s state farm was susceptible to the alpha-cypermethrin with a resistance ratio RR50 = 1.64 (95% CI: 0.2 to 12.6 ), all the other resistance tests conducted on moderate or high resistance Bassila and Kpinnou appear to host the most resistant samples. Moreover, a certain high variability of dose response relationship has been noticed with amitraz on the base of the higher slope of the related curves. Key words: Resistance ratio, distribution, Rhipicephalus microplus, acaricide, larval packet test (LPT), Benin