Research using film stimuli has shown that the strength of empathy-related responses toward nonhuman animals is related to the amount of phylogenetic similarity of the animals to humans. The present study aimed to develop and validate a new set of still images depicting humans and nonhuman animals (primates, quadruped mammals, birds, reptiles) in negative circumstances to provide greater control over variable image quality and contextual features found to be present in film stimuli. Following development of the stimulus set, participants (40 males, 46 females) with a mean age of 23.35 years made subjective ratings of empathy and other reactions to the images. The hypothesized effect of higher ratings of empathy for nonhuman animals that were more closely related to humans was supported. The same effect of phylogenetic similarity was also observed for ratings of arousal, dominance, interest, and pleasantness elicited by the images. Moreover, a clear effect of trait empathy emerged, consistent with the prediction that higher empathy ratings would be elicited from those higher in trait empathy. Familiarity, as assessed by level of involvement with the presence and care of animals, was also positively related to trait empathy. The findings validate a new methodology for the assessment of human empathy toward other humans and nonhuman species depicted in negative life situations. Furthermore, the findings demonstrate that human empathy generalizes toward other species according to perceived phylogenetic similarity.