| Summary: | There is a severe lack of information about molecular mechanisms of pesticide resistance in the rust-red flour beetle, a major pest destroying grains and flour across Nigeria, hindering evidence-based control. Here, we identified to the species level three populations of the red flour beetle from Kano, Nigeria, as <i>Tribolium castaneum</i> (Herbst 1797) and investigated the mechanism driving their insecticide resistance. The IRAC susceptibility bioassays established cypermethrin resistance, with LC<sub>50</sub>s of 4.35–5.46 mg/mL in the three populations, NNFM, R/Zaki and Yankaba. DDT and malathion resistance were observed in NNFM, with LC<sub>50</sub>s of 15.32 mg/mL and 3.71 mg/mL, respectively. High susceptibility was observed towards dichlorvos in all three populations with LC<sub>50</sub>s of 0.17–0.35 mg/mL. The synergist bioassay with piperonylbutoxide significantly restored cypermethrin susceptibility, with mortality increasing almost threefold, from 24.8% obtained with 1.5 mg/mL of cypermethrin to 63.3% in the synergised group (<i>p</i> = 0.013), suggesting a preeminent role of P450s. The two major knockdown resistance (<i>kdr</i>) mutations, T929I and L1014F, in the IIS4 and IIS6 fragments of the voltage-gated sodium channel were not detected in both cypermethrin-alive and cypermethrin-dead beetles, suggesting a lesser role of target-site insensitivity mechanisms. These findings highlight the need to explore alternative control tools for this pest and/or utilise synergists, such as piperonyl butoxide, as additional chemistries in pesticide formulations to improve their efficacy.
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