Multiple comorbidities related to arsenic exposure through drinking water continue to be public problems worldwide, principally in chronically exposed populations, such as those in the Comarca Lagunera (CL), Mexico. In addition, this relationship could be exacerbated by an early life exposure through the placenta and later through breast milk. This study conducted a comparative analysis of arsenic levels in multiple biological samples from pregnant women and their neonates in the CL and the comparison region, Saltillo. Total arsenic levels in placenta, breast milk, blood, and urine were measured in pregnant women and their neonates from rural areas of seven municipalities of the CL using atomic absorption spectrophotometry with hydride generation methodology. The average concentrations of tAs in drinking water were 47.7 µg/L and 0.05 µg/L in the exposed and non-exposed areas, respectively. Mean levels of tAs were 7.80 µg/kg, 77.04 µg/g-Cr, and 4.30 µg/L in placenta, blood, urine, and breast milk, respectively, in mothers, and 107.92 µg/g-Cr in neonates in the exposed group, which were significantly higher than those in the non-exposed area. High levels of urinary arsenic in neonates were maintained 4 days after birth, demonstrating an early arsenic exposure route through the placenta and breast milk. In addition, our study suggested that breastfeeding may reduce arsenic exposure in infants in arsenic-contaminated areas. Further studies are necessary to follow up on comorbidities later in life in neonates and to provide interventions in this region.