The Rio Grande chirping frog, Eleutherodactylus cystignathoides is native to southeastern Texas and northern Mexico. Over the past 40 years, populations of this species have been introduced into both the Houston and San Antonio regions. During this time all three populations have thrived under somewhat different environmental and living conditions. In southern Texas, this species can be found in its natural habitat of palm groves, thickets, ditches, and resacas. In urban areas, such as Houston and San Antonio, it appears on lawns and groundcover as well as in flowerbeds and bushes. Vocalization in amphibians is highly developed and frogs are known to produce a variety of sounds to attract mates, advertise territories, or express distress. The Rio Grande chirping frog vocalization is represented by both individual notes and trills (a rapid succession of note pulses). General, female frogs only respond to advertisement calls of their own species. Since the Houston, San Antonio, and southern Texas populations are isolated from one another, it is possible that variations in these vocalization patterns have developed. Differences in a male’s advertisement call from one population may not be recognized by a female in another population. Since females will not breed with a male she does not recognize as her own species, these variations may lead to reproductive isolation and speciation. We have studied the vocalizations of the Rio Grande Chirping frog from all three populations and variations in the three populations have been characterized. Population specific differences have been observed in frequency, amplitude, call duration, notes per call, notes per trill, trill length, and internote length. We are currently interested in characterizing Chirping frogs further to determine if their vocalizations may be affected by variations in temperature, humidity, barometric pressure, and time of the year, and if the differences in the population affects mating behavior.