It is estimated that 120 million people are infected by Ascaris lumbricoides in Latin America, but few genomic studies have been conducted. We tested protocols for DNA extraction to obtain an Ascaris sp. molecular diagnosis from human feces, with the emphasis on PCR specificity and sensitivity. Ascaris sp. was detected in 100% of positive fecal samples using physico-chemical DNA extraction and the cytb gene and ITS1 as molecular targets. The method was sufficiently sensitive to detect Ascaris sp. from one isolated egg or four eggs in a fecal sample. Regarding the PCR specificity, there was no cross-reactivity when applied to Trichuris trichiura-positive fecal samples or in Ascaris sp. samples also positive to T. trichiura or Enterobius vermicularis. The ITS1 sequence analysis revealed two genotypes among the sample: the G1 genotype, the most prevalent in humans, and a new genotype, G6, described for the first time in Brazilian samples.