Background Species within the Bemisia tabaci cryptic species complex can cause significant crop damage. We used high‐throughput amplicon sequencing to identify the species composition and resistance allele genotypes in field populations from cotton fields in Australia. For selected populations, the resistance phenotype was determined in bioassays and compared with sequencing data. Results A metabarcoding approach was used to analyse the species composition in 144 field populations collected between 2013 and 2021. Two mixed AUS I and MEAM1 populations were detected, whereas the remaining 142 populations consisted of MEAM1 only. High‐throughput sequencing of organophosphate and pyrethroid resistance gene amplicons showed that the organophosphate resistance allele F331W was fixed (> 99%) in all MEAM1 populations, whereas the pyrethroid resistance allele L925I in the voltage‐gated sodium channel gene was detected at varying frequencies [1.0%–7.0% (43 populations); 27.7% and 42.1% (two populations); 95%–97.5% (three populations)]. Neither organophosphate nor pyrethroid resistance alleles were detected in the AUS I populations. Pyrethroid bioassays of 85 MEAM1 field‐derived populations detected no resistance in 51 populations, whereas 32 populations showed low frequency resistance, and 2 populations were highly resistant. Conclusions We demonstrate that high‐throughput sequencing and bioassays are complementary approaches. The detection of target site mutations and the phenotypic provides a comprehensive analysis of the low‐level resistance to pyrethroids that is present in Australian cotton farms. By contrast, a limited survey of whitefly populations from horticulture found evidence of high‐level resistance against pyrethroids. Furthermore, we found that the F331W allele (linked to organophosphate resistance) is ubiquitous in Australian MEAM1. © 2022 Commonwealth of Australia. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

BACKGROUND: Bemisia tabaci is a globally significant agricultural pest including in Australia, where it exhibits resistance to numerous insecticides. With a recent label change, buprofezin (group 16), is now used for whitefly management in Australia. This study investigated resistance to pyriproxyfen (group 7C), spirotetramat (group 23) and buprofezin using bioassays and available molecular markers.RESULTS: Bioassay and selection testing of B. tabaci populations detected resistance to pyriproxyfen with resistance ratios ranging from 4.1 to 56. Resistance to spirotetramat was detected using bioassay, selection testing and sequencing techniques. In populations collected from cotton, the A2083V mutation was detected in three populations of 85 tested, at frequencies ≤4.1%, whereas in limited surveillance of populations from an intensive horticultural region the frequency was ≥75.8%. The baseline susceptibility of B. tabaci to buprofezin was determined from populations tested from 2019 to 2020, in which LC 50 values ranged from 0.61 to 10.75 mg L −1 . From the bioassay data, a discriminating dose of 200 mg L −1 was developed. Recent surveillance of 16 populations detected no evidence of resistance with 100% mortality recorded at doses ≤32 mg L −1 . A crossresistance study found no conclusive evidence of resistance to buprofezin in populations with high resistance to pyriproxyfen or spirotetramat.CONCLUSIONS: In Australian cotton, B. tabaci pest management is challenged by ongoing resistance to pyriproxyfen, while resistance to spirotetramat is an emerging issue. The addition of buprofezin provides a new mode-of-action for whitefly pest management, which will strengthen the existing insecticide resistance management strategy.