A Palmer amaranth biotype (CT-Res) with resistance to glyphosate was recently confirmed in a pumpkin field in Connecticut. However, the underlying mechanisms conferring glyphosate resistance in this biotype is not known. The main objectives of this research were 1) to determine the effect of plant height (10, 20, and 30 cm) on glyphosate resistance levels in CT-Res Palmer amaranth biotype, and 2) to investigate whether the target site–based mechanisms confer glyphosate resistance. To achieve these objectives, progeny seeds of the CT-Res biotype after two generations of recurrent selection with glyphosate (6,720 g ae ha−1) were used. Similarly, known glyphosate-susceptible Palmer amaranth biotypes from Kansas (KS-Sus) and Alabama (AL-Sus) were included. Results from greenhouse dose-response studies revealed that CT-Res Palmer amaranth biotype had 69-, 64-, and 54-fold resistance to glyphosate as compared with the KS-Sus biotype when treated at heights of 10, 20, and 30 cm, respectively. Sequence analysis of the EPSPS gene revealed no point mutations at the Pro106 and Thr102 residues in the CT-Res Palmer amaranth biotype. Quantitative polymerase chain reaction analysis revealed that the CT-Res biotype had 33 to 111 relative copies of the EPSPS gene compared with the AL-Sus biotype. All these results suggest that the EPSPS gene amplification endows a high level of glyphosate resistance in the GR Palmer amaranth biotype from Connecticut. Because of the lack of control with glyphosate, growers should adopt the use of effective alternative preemergence and postemergence herbicides in conjunction with other cultural and mechanical tactics to mitigate the further spread of GR Palmer amaranth in Connecticut.