The current study was carried out to assess 2 hypotheses: (1) cows differ in susceptibility to a subacute ruminal acidosis (SARA) challenge, and (2) the milk fatty acid (FA) pattern can be used to differentiate susceptible from nonsusceptible cows. For this, 2 consecutive experiments were performed. During experiment 1, the milk FA pattern was determined on 125 cows fed an increasing amount of concentrate during the first 4 wk in milk (WIM). The coefficient of variation of several SARA indicative milk FA (i.e., C15:0, C18:1 trans-10, C18:2 cis-9, trans-11, and C18:1 trans-10 to C18:1 trans-11 ratio) increased, indicating that cows reacted differently upon the concentrate build-up. A first grouping was based on the milk fat C18:1 trans-10 proportion in the third WIM. Fifteen cows with the highest proportion of the latter FA (HT10) and their counterparts with low C18:1 trans-10 and equal parity distribution (LT10) were compared, which revealed that milk fat content and milk fat to protein ratio were lower for the HT10 group. From each of the HT10 and LT10 groups, 5 animals were selected for experiment 2. The subselection of the HT10 group, referred to as HT10s, showed a high proportion of C18:1 trans-10 at 3 WIM (> 0.31 g/100 g of FA), a high level of C15:0 (on average >= 1.18 g/100 g of FA over the 4 WIM), and a sharp decrease of C18:1 trans-11 (Delta >= 0.25 g/100 g of FA during the 4 WIM). Their counterparts (LT10s) had a low milk fat C18:1 trans-10 proportion at 3 WIM (< 0.23 g/100 g of FA), an average C15:0 proportion of 0.99 g/100 g of FA or lower, and a rather stable C18:1 trans-11 proportion. The HT10s group was hypothesized to be more susceptible to a SARA challenge, achieved by increasing amounts of rapidly fermentable carbohydrates in experiment 2. The HT10s cows had a lower nadir, mean, and maximum reticulo-ruminal pH; longer period of reticulo-ruminal pH below 6.0; and higher daily reticulo-ruminal pH variation compared with LT10s cows. Throughout experiment 2, HT10s and LT10s cows differed in levels of SARA indicative milk FA. Five animals, including one LT10s and 4 HT10s cows, experienced SARA, defined as reticulo-ruminal pH < 6.0 for more than 360 min/d. These results indicate that it is possible to distinguish cows with different susceptibility to a SARA challenge within a herd by monitoring the milk FA composition when cows receive the same diet.