Anti-inflammatory treatment in early lactation has increased long-term milk yield in multiparous dairy cows, but mechanisms are unclear. Epigenetic changes alter chromatin structure and can affect gene expression patterns long-term, and tissues undergoing development and cellular differentiation are more susceptible to epigenetic changes. As metabolic changes in early lactation involve multiple tissues, we examined tissue samples from 2 different studies for changes to global methylation (GM) as a potential mechanism for long-term effects of anti-inflammatory treatment. In study 1, 16 cows of parity ≥ 3 were alternately assigned to treatment at parturition and were drenched orally with sodium salicylate (SS; 125 g/d) or water (CON) once daily for 3 d following parturition, beginning ~24 h postpartum. Mammary tissue was collected on d 1 (before treatment), d 4, and d 45 of lactation. In study 2, 17 multiparous cows were similarly enrolled at calving and administered either SS (2 g/L) or molasses carrier (CON) via the drinking water for 5 d after parturition. Samples were collected on d 5 from adipose (tailhead), liver, and muscle tissue (longissimus dorsi lumborum). DNA was isolated and GM was assessed with a 5-methylcytosine ELISA. Mixed models were used to assess GM; contrasts compared treatment effects within day. No differences were detected in mammary GM on d 1 (5.6 vs. 5.8 ± 0.7 for SS vs. CON; P = 0.78), but SS increased GM on d 4 compared with CON (5.8 vs 4.1 ± 0.6%; P = 0.04). Although treatment means on d 45 were similar to d 4, mammary GM differences were not detected (5.6 vs. 4.5 ± 0.6%; P = 0.21). No treatment differences were detected for GM in liver, adipose, or muscle tissues (P ≥ 0.33). Although GM does not identify regions of DNA or downstream functions affected, an observed tendency for GM to decrease from d 1 to d 4 for CON (5.6 vs 4.0 ± 0.6%; P = 0.07) supports the occurrence of epigenetic changes during the onset of lactation, and the possibility that SS may elicit long-term effects via alterations to methylation patterns.