Abstract: We established families based on 3 different geographic populations of wild Penaeus monodon from Africa (F), Thailand (T) and Indonesia (Y), and compared their tolerance to ammonia-N by a 96-hour acute toxicity test. The results show that the mortalities of those families were 15.56%~100% under high ammonia-N (30.0 mg·L^-1) stress in 96 h, and very significant difference was found among different families (P < 0.01). Seven families (mortalities are lower 30%) had high tolerance to ammonia-N; twenty-nine families (mortalities are 30%~60%) had medium tolerance; nine families (mortalities are higher than 60%) had low tolerance. The tolerance to ammonia-N of families established based on different male and female parents from high to low is Africa, Indonesia and Thailand; that of different mate combinations from high to low is F♀×F♂, Y♀×Y♂, Y♀×T♂, T♀×Y♂ and T ♀×T♂. The tolerances to ammonia-N of the hybrid combinations between Indonesia and Thailand populations (T♀ × Y♂ and Y♀ × T♂) were 1.98% and 19.8%, respectively. The study provides references for the selective breeding of P.monodon lines with high tolerance to ammonia-N.