This study aimed to investigate whether breed, sex, and age affected temperament differently (more or less neophobic) in mature horses during a novel object test. The study included Jeju crossbred (n = 12, age = 9.42 ± 4.57 y), Thoroughbred (n = 15, age = 10.73 ± 3.09 y), and Warmblood horses (n = 12, age = 13.08 ± 3.55 y) with the females (n = 22, age = 11.36 ± 4.24 y) and geldings (n = 17, age = 10.65 ± 3.66 y). Jeju crossbreds (Jeju horse × Thoroughbred) are valuable considering their popular usage in South Korea, but limited studies have explored temperament of Jeju crossbred horses. A trained experimenter touched the left side of the neck with a white plastic bag (novel object). The test ended when the horse stopped escape response and heart rate dropped to baseline. Behavioral score and escape duration were measured as behavioral variables. Multiple variables related to heart rate (HR) and heart rate variability (HRV) were measured to reflect emotional state. These included basal HR (BHR), maximum HR (MHR), delay to reach maximum heart rate (Time to MHR), standard deviation of beat-to-beat intervals (SDNN), root mean square of successive differences (RMSSD), and ratio of low to high frequency components of a continuous series of heartbeats (LF/HF). Statistics revealed that Thoroughbreds had significantly higher behavioral scores, and lower RMSSD than Jeju crossbreds (p < 0.05), suggesting greater excitement and fear to the novel object in Thoroughbreds. None of the behavioral or cardiac parameters exhibited sex differences (p < 0.05). Age was negatively correlated with SDNN and RMSSD (p < 0.05), indicating that older horses felt more anxiety to the novelty than younger horses. Thoroughbreds and females had distinct correlations between behavioral and HRV variables in comparison with other groups (p < 0.05), implying that escape duration might be a good indicator of stress, especially in these two groups. These results are expected to improve equine welfare, safety and utility, by providing insights into the temperament of particular horse groups, to better match reactivity levels with specific functions.