African Animal Trypanosomosis is threatening the agricultural production and cattle breeding more severely than any other livestock disease in the continent, even more since the advent of drug resistance. A longitudinal study was conducted from November 2012 to May 2013 in the Ghibe valley to evaluate diminazene aceturate (DA) resistance and assess livestock owner's perception of trypanocidal drug use. Four Peasant Associations (PAs) were purposively selected and the cattle randomly sampled in each PAs. At the beginning of the study (t0), 106 bovines positive for trypanosomes by the haematocrit centrifugation technique (HCT) and 119 negative control animals were recruited for six months follow-up using HCT, 18S-PCR-RFLP, DpnII-PCR-RFLP and microsatellite analysis. Prevalence of trypanosomosis was 18.1% based on the HCT technique and the mean PCV value was 23.6±5.1% for the 587 sampled cattle. Out of the 106 HCT positive, 64 (60.4%) were positive for the presence of trypanosomes using the 18S-PCR-RFLP. Species detection showed 38 (59.4%) Trypanosoma congolense savannah, 18 (28.1%) Trypanosoma vivax, 5 (7.8%) Trypanosoma theileri and 3 (4.7%) T. congolense Kilifi. Among the T. congolense savannah samples, 31 (81.6%) showed a DA resistant RFLP profile, 2 (5.3%) a mixed profile and 5 did not amplify using the DpnII-PCR-RFLP. A positive HCT had a significant effect on PCV (p<0.001) with the mean PCV value equal to 24.4±0.2% in the absence of trypanosomes and to 20.9±0.3% in the presence of trypanosomes. PCV increased significantly (p<0.001) with 4.4±0.5% one month after treatment. All T. congolense savannah type were analyzed using microsatellite markers TCM1, TCM3 and TCM4. The main events were new infections (40.0%) and relapses (37.5%) with cures lagging at 22.5%. In 10 purposively selected PAs a semi-structured questionnaire was used. The average herd size was the highest in Abelti PA (6.7±1.8 TLU) and the mean herd size was statistically different (p=0.01) in the 10 PAs. Trypanosomosis was designated as the main disease affecting cattle by 97% of the respondents. DA was used by 95.5% of the farmers though more than half of them (51.9%) were not familiar with isometamidium (ISM). There was a trend to overdose young small animals and to underdose large ones. Oxen were treated very frequently (nearly 20 times/year) and calves almost never. To improve the situation in the Ghibe valley, extension messages should be delivered to promote a rational drug use, improved livestock management and the application of strategic vector control methods.