Uterine leiomyoma is the most frequent pelvic tumor found in female genital tract. Some studies have suggested an association between single nucleotide polymorphisms (SNPs) in estrogen receptors genes with susceptibility in developing uterine leiomyoma. In this work, we estimated the frequency of two SNPs: one located in the intron 1 (rs9322331) and other in the exon 1 (rs17847075) of the estrogen receptor alpha (ESR1) gene in 125 women with uterine leiomyoma and 125 healthy women. To do this we used a PCR-RFLP method with MspI and HaeIII restriction enzymes to respectively detect C/T SNPs in the intron 1 and in the exon 1 of ESR1. To our knowledge this is the first study aimed to investigate the association of ESR1 SNPs with the risk of developing uterine leiomyoma in Brazilian women. Our results showed that the allele frequencies of the exon 1 and the intron 1 of the ESR1 gene did not differ between cases and controls (P = 0.325 and 0.175, respectively). Furthermore, our findings provided little support for the association of these SNPs on ESR1 with leiomyoma. However, we found that the SNP in the intron 1 of the ESR1 gene was underrepresented in the Brazilian female population.