Susceptability of Ascaridia galli to benzimidazole (BZ) was investigated using faecal egg count reduction test (FECRT), in ovo larval development test (LDT) and genetic markers (mutations at codons 167, 198 and 200 of β-tubulin gene). Six flocks (F1−F6) of a commercial laying hen farm with different number of exposure to BZ were recruited. The FECR was calculated by analyzing individual faeces (F1, F2, F4 and F5) before and 10 days after treatment. The LDT was performed on parasite eggs from pooled samples from F1 to F6 and LC50 and LC99 were calculated. DNA was extracted from 120 worms and sequenced for β-tubulin gene. In all flocks, the FECRs were above 95% (lower CI above 90%). No significant difference was observed (p > 0·05) among obtained LC50 (F1/F4 and F2/F5 vs F3/F6) in the LDT. However, LC50 and LC99 were higher than suggested values for declaration of resistance in other nematode species. No variation was observed in codon positions involved in BZ resistance. Overall, our results indicated lack of evidence of resistance to BZ in A. galli. More research is needed to confirm these results and to further optimize the existing tools for detection and monitoring of anthelmintic resistance in A. galli.