Portugal has the fourth highest tuberculosis (TB) incidence rate in the European Union (EU). Thirty-nine percent of all cases originate in Lisbon Health Region. Portugal also presents high levels of multidrug-resistant tuberculosis (MDR-TB) (1.5%, primary rate and 2.4%, in retreatment cases). In the present study we have characterized 58 MDR-TB clinical isolates by: (i) determining the resistance profile to first- and second-line drugs used in the treatment of tuberculosis; (ii) genotyping all isolates by MIRU-VNTR; (iii) analyzing mutations conferring resistance to isoniazid, rifampicin, streptomycin, and ethambutol, in katG, mabA-inhA, rpoB, rpsL, rrs, and pncA genes. We have therefore established the prevalence of the most common mutations associated with drug resistance in the Lisbon Health Region: C-15T in mabA-inhA for isoniazid; S531L in rpoB for rifampicin; K43R in rpsL for streptomycin; and V125G in pncA for pyrazinamide. By genotyping all isolates and combining with the mutational results, we were able to assess the isolates' genetic relatedness and determine possible transmission events. Strains belonging to family Lisboa, characterized several years ago, are still responsible for the majority of the MDR-TB. Even more alarming is the high prevalence of extensive drug-resistant tuberculosis (XDR-TB) among the MDR-TB isolates, which was found to be 53%. The TB status in Portugal therefore requires urgent attention to contain the strains continuously responsible for MDR-TB and now, XDR-TB.