Invasive disease caused by Haemophilus influenzae serotype b results in high rates of morbidity and mortality among children. In 1994, the Microbiology Group at the Instituto Nacional de Salud (Colombia) initiated a program to detect antimicrobial resistance in H. influenzae. Invasive isolates were collected by hospitals and public health laboratories as part of surveillance programs for acute respiratory infections and acute bacterial meningitis. To determine the evolution of serotypes and antimicrobial resistance patterns, invasive H. influenzae isolates collected from 1994 to 2002 were compared, and the impact of Hib conjugated vaccine in Colombia was reassessed. The analysis included 683 isolates, 379 (55.5%) were recovered from male patients, 370 (54.2%) from children under one year, 227 (33.2%) from children aged 1 to 5, 19 (2.8%) from children aged 6 to 14, and 38 (5.6%) from children over 14 years; 29 (4.2%) with no age data. Clinical classification recorded 493 (72.2%) of the samples were from patients with meningitis, 181 (26.5%) with pneumonia, and 9 (0.9%) with other diseases. Eighty five percent of isolates corresponded to H. influenzae serotype b, 12.9% were non capsular, and 2.0% corresponded to other serotypes (10 a, 1 d, 1 e and 2 f). Of the total number of isolates, 12.0% produced beta lactamase, 13.9% were resistant to ampicillin, 12.7% to trimethoprim sulfamethoxazole (SXT), 5.4% to chloramphenicol, 1% to cefuroxime. All isolates were susceptible to ceftriaxone. During the 10-year period, resistance to SXT increased from 5% to 13%. A significant decrease in meningitis cases was detected among children under one-year old and in the 1 to 5 age group. Before introducing the vaccine, an annual average of 43 and 23 isolates for each of these groups were received. During 2002, 10 and 6 isolates, respectively, were received for each group. Surveillance of invasive H. influenzae isolates has allowed the evaluation of Hib vaccine impact, as well as the detection of an increase of non-capsular isolates, and changes in resistance patterns.