The objective of this study was to assess antibiotic resistance and the molecular epidemiology of shigella isolates from a case-control study of diarrhoea, conducted from 2007 to 2012 in children aged less than 5 years in Manhiça district, southern Mozambique. All isolates were tested for antimicrobial susceptibility using the disc diffusion method. Polymerase chain reaction was used to detect different molecular mechanisms of antibiotic resistance. Serotyping was performed using specific antisera. The clonal relationship of Shigella flexneri and Shigella sonnei was assessed by pulsed-field gel electrophoresis (PFGE). Of the 67 shigella isolates analysed, 59 were diarrhoeal cases and eight were controls. S. flexneri (70.1%; 47/67) was the most common species, followed by S. sonnei (23.9%; 16/67). The most prevalent S. flexneri serotypes were 2a (38.3%; 18/47), 6 (19.2%; 9/47) and 1b (14.9%; 7/47). High rates of antimicrobial resistance were observed for trimethoprim-sulfametoxazole (92.5%; 62/67), tetracycline (68.7%; 46/67), chloramphenicol (53.7%; 36/67) and ampicillin (50.7%; 34/67). Multi-drug resistance (MDR) was present in 55.2% (37/67) of the isolates and was associated with a case fatality rate of 8.1% (3/37). PFGE revealed 22 clones (16 S. flexneri and 6 S. sonnei), among which P1 (31.9%; 15/47), P9 (17%; 8/47) and P2 (10.6%; 5/47) were the most prevalent clones of S. flexneri. In conclusion, S. flexneri was the most prevalent species, with MDR isolates mainly belonging to three specific clones (P1, P9 and P2). The case fatality rate observed among MDR isolates is a matter of concern, indicating the need for appropriate treatment.