Objective. To assess the relationship between the presence of mixed infection of Helicobacter pylori and both antimicrobial susceptibility and virulence markers. Material and methods. Thirty-six patients with H. pylori infection were included in the study. Three colonies were selected from each positive biopsy sample collected from each host for a total of 108 H. pylori strains. The genetic variability was evaluated through the amplified fragment length polymorphism (AFLP) analysis; the antibiotic susceptibility to amoxicillin, clarithromycin, moxifloxacin, rifabutin and tinidazole was determined using the minimum inhibitory concentrations (MICs) with the agar dilution method. Moreover, the vacA, cagA, iceA and bab- status were detected by polymerase chain reaction (PCR). Results. There was a strong connection between mixed H. pylori infection and antimicrobial resistance. In particular,H. pylori strains with genetic variability, in the same host, expressed more resistance to clarithromycin, moxifloxacin and tinidazole than that expressed in strains with a unique genetic host pattern. VacA s1m1/s1m2 genotypes were found in 70% of strains isolated in mixed infection, whereas the same allelic combinations were found in 42% of strains, isolated in single infection. The cagA+ status prevailed both in patients with mixed (97% ) and in those with single infection (85% )without significant differences. The iceA1 status was more commonly found in patients with mixed infection, whereas the bab- status was significantly prevalent in single H. pylori infection. Conclusions. Mixed H. pylori infection harbouring in one patient is significantly related to strains that are more resistant to antibiotics and with a more virulent genotype (vacA s1m1/s1m2, cagA, iceA1) than strains responsible for single infection. Copyright ? 2006 Elsevier B. V Amsterdam. All rights reserved. No part of this publication may be reproduced, stored in a retrieval system or transmitted, in any form or by any means, electronic, electrostatic, magnetic tape, mechanical, photocopying, recording, or otherwise, without prior written permission of the Publisher, Elsevier B.V., Radarweg 29, 1043NX Amsterdam, The Netherlands