Hemophilia A (HA) is an X-linked coagulation disorder caused by a deficiency of the clotting factor VIII (FVIII). Diagnosis of HA is based on the dosage of plasmatic residual activity of FVIII. Brazilian HA patients are poorly molecular diagnosed and there is a lack of genetic data about the FVIII gene variants in the country. The aim of this study was to perform molecular diagnosis of patients with HA treated at Fundação Hemominas using next generation sequencing (NGS). Patients were invited to participate when they attended appointments and/or examinations at Fundação Hemominas. A custom Illumina panel containing the coding region of the F8, F9, and FVW genes was used to prepare the libraries, and the run was performed on the MiSeq sequencer (Illumina). Reads, were aligned with the reference genome GRCh38.p14 and variants analyzes were performed using dbSnp, ClinVar, LOVD (Leiden Open Variation Database), EAHAD (EAHAD Coagulation Factor Variant Databases), and varsome. Thirty-six patients were included (25 with severe HA, 8 with moderate HA, and 3 with mild HA), five of whom had low gene coverage impairing analysis. In one patient, no variants were found in the F8 gene but only in the VWF gene. In the remaining 30 patients, 38 variants were identified in the F8 gene, classified as follows: 10 probably benign, nine probably pathogenic, nine pathogenic/probably pathogenic, four benign/probably pathogenic, three not reported, two benign/probably benign, and one variant of unclear significance (VUS). Pathogenic or likely pathogenic variants were identified in 25 patients (17 severe, six moderate, and two mild). Among the pathogenic or probably pathogenic variants found, six are new (chrX:154993148; chrX:154954014; chrX:154931503; chrX:154929482; chrX:154930824; chrX:154837665) and 12 have been described previously (rs2073186194, rs1331801498, rs137852357, rs1168154820, rs137852358, rs137852367, rs1455987570, rs387906432, rs1367630608, rs1432162307, rs137852442, rs111033614). Three variants (chrX:154993148, rs137852367, and rs1455987570) are located in the plastocyanin-like 1 and F5/8 type A1 domains of the FVIII protein and cause alterations in the cDNA c.389G>A, c.566C>G, and c. 269T>G, respectively, as well as alterations in the proteins p.Gly130Glu, p.Ser189*, and p.Leu90Arg. Variant chrX:154954014 is located in the plastocyanin-like4 and F5/8 typeA2 domains of the FVIII protein and causes alterations in cDNA c.1779delC and protein p.Leu594fs. Five variants chrX:154931503, chrX:154930824, rs2073186194, rs137852357 and rs1168154820 are located in the B domain of the FVIII protein and each causes alterations in cDNA c.2287C>T, c.2962_2963insGT, c.3629delA, c.6496C>T, and c.3982C>T and protein changes in p.Gln763*, p.Gly989fs, p.Ile1213fs, p.Arg2166*, and p.Gln1328*. Two variants (rs1432162307 and rs137852442) are located in the plastocyanin-like5 and F5/8typeA3 domains of the FVIII protein and cause changes in cDNA c.5178G>C and c.5399G>A, respectively, and changes in protein p.Trp1726Cys and p.Arg1800His. The rs1367630608 variant is located in the plastocyanin-like6 andF5/8 typeA3 domains of the FVIII protein and causes alterations in cDNA c.5981T>G and protein p.Leu1994Arg. Two variants (chrX:154837665 and rs137852358) are located in the F5/8typeC2 domain of the FVIII protein and cause changes in cDNA c.6988C>T and c.6683G>A, respectively, and changes in protein p.Gln2330* and p.Arg2228Gln. Variants rs387906432, rs111033614, and rs1331801498 are not in any domain of the FVIII protein but each cause changes in cDNA c.43C>T, c.5123G>A, and c.6575-1G>A and in protein p.Arg15* and p.Arg1708His. In this context, molecular diagnosis proved to be important to determine which variants are responsible for the disease in patients.