Circulating nucleic acids (CNAs) are free-floating, cell-free DNA and RNA molecules in the circulation of healthy and diseased humans and animals. The aim of this study was to identify differences in CNA distribution in serum samples from multiparous pregnant (n = 24) and nonpregnant (n = 16) dairy cows at different days of gestation (Days 0, 20, and 40). A modified serial analysis of gene expression procedure was used to generate concatemerized short sequence tags from isolated serum DNA. A total of 6.1 × 10⁶ tags were recovered from analyzed samples (n = 40). Significant differences between the pregnant and nonpregnant groups were detected in chromosomal regions, protein-coding sequences, and single genes (P < 0.05). Approximately 23% (1.4 × 10⁶ tags) of the total sequence pool were present exclusively in the analyzed serum samples of pregnant cows. Of these tag sequences, seven originated from genomic regions and 13 from repetitive elements. Comparative BLAST analysis identified the repetitive tags as BovB (non-long terminal repeat retrotransposons/long interspersed nuclear elements), Art2A, BovA2, and Bov-tA2 (short interspersed nuclear elements). To our knowledge, this is the first study to comprehensively characterize the circulating, cell-free DNA profile in sera from pregnant and nonpregnant cows across early gestation.