The aim of this study was to assess the potential horizontal transfer of genetic traits for antibiotic resistance between lactobacilli isolated from the chicken gut, both in vitro and in vivo. Thirty-seven Lactobacillus spp. strains isolated from the gizzard, small and large intestines and caeca of free-range broiler chickens showed multi-drug resistance as assessed by disc diffusion assays. The minimum inhibitory concentration (MIC) for vancomycin, tetracycline, erythromycin and chloramphenicol was determined in De Man, Rogosa and Sharpe broth in a microplate assay. Almost all the lactobacilli isolates were resistant to vancomycin (except strains belonging to the Lactobacillus acidophilus group) and to tetracycline (MIC≥128 μg/ml). Only five strains were resistant to erythromycin, and six to chloramphenicol. The transfer rate in filter mating experiments performed using L. acidophilus strain 4M14E (Em^R), Lactobacillus vaginalis strain 5M14E (Cm^R), Lactobacillus salivarius strain 5C14C (Em^R), and the 4G14L and 3C14C strains of Lactobacillus reuteri (Cm^R) showed a frequency of approximately 1×10⁴ cfu/ml of double-resistant transconjugants for the different combinations. The exception was the L. salivarius 5C14C (Em^R) and L. vaginalis 5M14E (Cm^R) mating combination, which produced no transconjugants. In vivo experiments performed in gnotobiotic mice by mating L. acidophilus 4M14E (Em^R) with L. reuteri 3C14C (Cm^R), L. reuteri 4G14L (Cm^R) or L. vaginalis 5M14E (Cm^R) resulted in transconjugants at 3.95±0.29, 3.16±0.33, and 4.55±1.52 log₁₀ cfu/g of faeces, respectively. Taken together, these data suggest that genetic exchange may occur between native bacterial strains within the gastrointestinal tract of chickens, which might maintain a dynamic gene pool conferring antibiotic resistance upon indigenous microbiota components, even in the absence of the pathogens. This possibility must be taken into account as a complementary criterion when lactobacilli are screened for probiotic use.