As a result of advances in analytical methodologies over recent years, novel endogenous anabolic-androgenic steroids (AAS) have been identified and detected in equine samples, especially in colts due to naturally high steroid concentrations and high aromatase activity. Therefore, to gain a better understanding of other potentially endogenous AAS in equine samples and to ensure effective doping control, a sensitive analytical method for detection of 14 selected steroids in equine urine was developed and subsequently applied to analysis of urine population samples from each gender (50 geldings, 50 mares and 36 colts), urine samples from a pregnant mare and selected administration studies (DHEA/pregnenolone, testosterone esters, boldenone undecylenate and nandrolone laurate). A number of steroids were detected for the first time in these samples, including the following in one or more genders: dienolone, dienedione, 19-norandrostenedione, 6-oxo, 6α-OH-androstenedione, formestane, 4-OH-testosterone and 7-keto-DHEA. Further studies are required to assess the potential microbial formation of these steroids and to establish approaches to differentiate their endogenous presence from administrations.