Several studies have reported gross composition differences between organic and conventional milk; however, most studies have not considered other factors such as breed and diet ingredients, which are known to influence milk composition. Thus, this study aimed to provide a detailed characterization of Holstein-Friesian cow milk from organic (ORG) and conventional (CONV) herds with similar diet ingredients and in the same geographic area. Bulk milk samples (n = 225) of 12 ORG and 12 CONV farms were collected from September 2019 to August 2020. Farms were located in Northern Italy, included corn (meal, silage, or both) in the lactating diets, and had similar management conditions, but ORG herds spent a period on pasture. Factors affecting milk composition were tested using a linear mixed model, which included calendar month, farming system (ORG and CONV), and their interactions as fixed effects, and farm nested within farming system as random effect. Results showed that total fat, lactose, vitamin E, and AA did not significantly differ between farming systems. Total protein and casein contents were significantly lower in ORG than CONV herds, and somatic cell score (SCS) was greater in ORG than CONV. Among minerals, differences were observed for Fe, K, Mg, and S in some months, being lower in ORG than CONV for K, Mg, and S and greater or lower for Fe depending on the month. Among fatty acid (FA) groups, index, and ratios, only polyunsaturated FA and n-3 FA tended to be greater in ORG than CONV, and cis-FA were greater in ORG than CONV during October. Among the most abundant individual FA, only C16: 1n -9 differed, being lower in ORG than CONV. The calendar month (and hence seasonal feed ration) was significant for milk gross composition, SCS, vitamin E, mineral profile (except for Mo, Sr, and Zn), AA profile, FA groups (except for medium-chain FA), FA index and ratios, and individual FA (except C16:0). We conclude that the overall milk composition was quite similar between the 2 farming systems. This could be related to the similarity of the selected farms, the Holstein-Friesian breed, and generally high level of intensity in both farming systems.