Resistance to glufosinate has been confirmed in glyphosate-resistant Italian ryegrass populations collected in hazelnut orchards in Oregon. Dose–response, ammonia accumulation, and enzyme activity studies were conducted to test the sensitivity of three glyphosate-resistant and three susceptible Italian ryegrass populations to glufosinate. The glufosinate rates required to reduce the growth by 50% (GR50) were 0.15, 0.18, and 0.21 for the control populations C1, C2, and C3, respectively, whereas for the resistant populations OR1, OR2, and OR3, the GR50 values were 0.49, 0.42, and 0.40 kg ai ha−1, respectively, exhibiting an average resistance index of 2.4. The same trend was observed in ammonia accumulation studies between 48 and 96 h after glufosinate treatment where the susceptible populations accumulated on average two times more ammonia than the resistant populations. The glufosinate concentration required to reduce the glutamine synthetase enzyme activity by 50% (I50) was not different for the resistant and susceptible populations. The I50s ranged from 3.1 to 3.6 µM for the resistant populations and from 3.7 to 4.3 µM for the susceptible populations; therefore, an insensitive target site is not responsible for the glufosinate resistance.