Haloxyfop is one of two acetyl-coenzyme A carboxylase (Accase) inhibitors that is recommended for controlling Poa annua. We have characterised a population of P. annua that had developed resistance to haloxyfop. This resistant population was found to be almost 20 times less sensitive to haloxyfop than a susceptible population based on percentage survival of individuals in two dose-response experiments. However, the haloxyfop-resistant population was still susceptible to clethodim. pre-treatment of resistant individuals with a cytochrome P450 inhibitor, malathion, did not change the sensitivity level of the resistant plants to haloxyfop, suggesting that a non-target site mechanism of resistance involving enhanced metabolism, was not responsible for this resistance in P. annua. Gene sequencing showed that a target site mutation at position 2041, which replaced isoleucine with threonine in the carboxyltransferase (ct) domain of the Accase enzyme, was associated with resistance to haloxyfop in the resistant population. An evaluation of the 3-D structure of the CT domain suggested that, unlike Asn-2041, which is the most common mutation at this position reported to date, Thr-2041 does not change the conformational structure of the CT domain. This is the first study investigating the molecular mechanism involved with haloxyfop resistance in P. annua. Poa annua, an annual C 3 weedy grass in both agricultural and urban systems 1,2 , is one of the world's most widely distributed plant species 2. It is particularly problematic in turf grass, where it is highly tolerant of disturbance and has high fecundity 3. The occurrence of P. annua is often associated with human activities, and is one of the weedy grass species that is often found in seedbanks 4. P. annua is a prolific annual weed with a very short life 2. Notably, it can produce seed throughout most of the year 1. Collectively, these features predispose P. annua to herbicide resistance, since, at any one time, a large number of individuals will experience selection pressure under intense herbicide use 5. P. annua is an allotetraploid (2n = 4 × = 28) species, originating from the hybridization of P. supina as the male parent and P. infirma as the female parent 6. Cultural practices that can help desirable species compete well with P. annua can be used for management of this weedy grass species in turf grasses 7 ; i.e. in an effort to stop it re-establishing from seed after it completes its life cycle. Techniques such as soil aerification, fertilization management and modifying irrigation regimens are useful cultural practices for P. annua management in turf 8. However, cultural techniques should be part of a management package 9 , as these techniques do not result in complete control of P. annua in turf. Indeed, application of herbicides is desirable for complete management of this weed species in turf 10. There are, however, only a limited number of herbicides that can selectively remove P. annua from turf grasses 11. Hence, the best strategy to manage P. annua in tur...