The reason for undertaking this study was to investigate soft tissue response to high-intensity laser therapy (HILT) by measuring changes in skin surface temperature and longissimus dorsi muscle tone in the thoracolumbar back area in Thoroughbreds with back pain and diagnosed with and without Kissing Spines Syndrome (KSS). Thoroughbreds aged 3-4 years with clinically presented back pain underwent a radiological examination (to assess a lack or presence of KSS) and longissimus dorsi muscle palpation (to assess muscle tone and pain degree). The subjects were divided into two groups, those with KSS (n = 10) and those without KSS (n = 10). A single HILT treatment on the longissimus dorsi muscle, on the left side, was performed. Thermographic examination and palpation were repeated before and after HILT to assess changes in skin surface temperature and muscle pain response. In both groups, HILT caused a significant increase in skin surface temperature of 2.5 °C on average and a palpation score reduction of 1.5 degrees on average (p = 0.005 for both measurements), without differences in any outcome measures between the groups. Furthermore, the correlation between changes in the average skin surface temperature and the average palpation scores in horses with and without KSS were negative (rho = 0.071 and r = -0.180, respectively; p > 0.05). The results of the present study are encouraging, but further studies with larger samples, a longer follow-up period and comparisons with placebo control groups are needed to draw a more valid conclusion.