Interspecific hybridization has a much greater effect than chromosome doubling on gene expression; however, the associations between homeologous gene expression changes and polyhaploidization had rarely been addressed. In this study, cDNA-single strand conformation polymorphism analysis was applied to measure the expression of 30 homeologous transcripts in naturally occurring haploid (ABD, 2n = 21) and its polyploid maternal parent Yumai 21A (AABBDD, 2n = 42) in wheat. Only one gene (TC251989) showed preferentially silenced homoeoalleles in haploids. Further analyses of 24 single-copy genes known to be silenced in the root and/or leaf also found no evidence of homeologous silencing in 1-month-old haploids and two ESTs (BF484100 and BF473379) exhibit different expression patterns between 4-month-old haploids and hexaploids. Global analysis of the gene expression patterns using the Affymetrix GeneChip showed that of the 55,052 genes probed, only about 0.11% in the shoots and 0.25% in the roots were activated by polyhaploidization. The results demonstrate that activation and silencing of homoeoalleles were not widespread in haploid seedlings.