There is a great deal of evidence that points to the association of the tumor necrosis factor-alpha (TNF-alpha) gene as a common genetic factor in the pathogenesis of diseases that are caused by inflammatory and/or autoimmune etiologies. Two single nucleotide polymorphisms (SNPs) identified in the TNF-alpha promoter region have been associated with disease susceptibility and severity. We investigated whether -308G/A and -238G/A TNF-alpha polymorphisms were associated with asthma, systemic lupus erythematosus (SLE), and juvenile rheumatoid arthritis (JRA) in a pediatric Mexican population. In a case-control study of 725 patients (asthma: 226, JRA: 171, and SLE: 328) and 400 control subjects, the participants were analyzed using the allelic discrimination technique. The genotype distribution of both TNF-alpha polymorphisms was in Hardy-Weinberg equilibrium in each group. However, there were significant differences in the allele frequency of TNF-alpha-308A between the patients and the healthy controls. This allele was detected in 2.9% of the controls, 6.0% of asthmatic and JRA patients (p = 0.002 and p = 0.0086), and 6.7% of SLE patients (p = 0.00049); statistical significance was maintained after ancestry stratification (asthma: p = 0.0143, JRA: p = 0.0083, and SLE: p = 0.0026). Stratification by gender showed that the risk for the -308A allele in asthma and JRA was greater in females (OR = 4.16, p = 0.0008 and OR = 4.4, p = 0.0002, respectively). The TNF-alpha -238A allele showed an association only with JRA in males (OR = 2.89, p = 0.004). These results support the concept that the TNF-alpha gene is a genetic risk factor for asthma, SLE, and JRA in the pediatric Mexican population.