Adenoviruses have been implicated in childhood cancers, primarily leukemia, yet prior neonatal investigations have rarely examined other pediatric tumor types. This study evaluated whether adenoviral early region (E1A) sequences can be detected in archival neonatal Guthrie cards from children who later developed diverse pediatric tumors and in corresponding paraffin-embedded tissues. DNA extraction was optimized for long-stored dried blood spots, and PCR conditions were refined for both Guthrie card and paraffin-derived DNA. Adenoviral E1A was analyzed using conventional and nested PCR, and sequencing of representative amplicons confirmed correspondence to human adenovirus species C. E1A PCR positivity was found in 43% of Guthrie cards from cases (n = 54) and 34% of controls (n = 32), and in 41% of tumor tissues (n = 75) compared with 5% of non-tumor paraffin controls (n = 20). Detection occurred across multiple tumor categories without a clear association with tumor type. Partial concordance was observed between paired neonatal and tumor samples, and cytomegalovirus markers were detected in a subset of E1A-positive specimens. These findings confirm the suitability of Guthrie cards for retrospective viral DNA detection and extend previous leukemia-focused neonatal studies to broader pediatric tumors. The data suggest a potential association between birth-stage adenoviral detection and childhood cancer, though a causal link remains unproven and requires further longitudinal investigation.